M Savy

S Fortin

Y Kameli

S Renault

C Couderc

A Gamli

K Amouzou

M L Perenze

Y Martin-Prevel

& S Fortin

& S Renault

& C Couderc

& A Gamli

& K Amouzou

Martin-Prevel

Impact of a food voucher program in alleviating household food insecurity in two cities in Senegal during a food price crisis

Keywords: Quasi-experimental evaluation, Food vouchers, Dietary diversity, Impact evaluation, Urban food security, Senegal, Sub-Saharan Africa

Impact of a food voucher program in alleviating household food insecurity in two cities in Senegal during

a food price crisis

Introduction

Significant progress has been made in reducing global food insecurity rates over the past 20 years. According to the Food and Agriculture Organisation (FAO), the estimated worldwide prevalence of undernourishment has declined from 18.6% in 1990-92 to 10.6% in 2015, but this prevalence is stagnating since then [START_REF] Fao | The State of Food Security and Nutrition in the World[END_REF]). An intolerable number of people still suffer from food and nutrition insecurity, in particular in Southern Asia and sub-Saharan Africa where progress is slow. As household food insecurity has been associated with either stunting in children under 5 or with obesity in school-age children, adolescents and adults, depending on contexts, it is essential to continue the efforts [START_REF] Fao | The State of Food Security and Nutrition in the World[END_REF].

Social protection programs are thought to have contributed to the global effort. Such programs have become a mainstay in social transfers and poverty reduction strategies throughout Latin America, and are increasingly being implemented in Sub-Saharan Africa [START_REF] Garcia | The Cash Dividend -The Rise of Cash Transfer Programs in Sub-Saharan Africa[END_REF]. Among them, cash transfer (CT) programs -which imply giving money in the form of cash, checks, money orders or vouchers, and which can either be conditional or unconditional -are becoming increasingly popular worldwide, probably due to their versatility. Numerous large-scale conditional cash transfer (CCT) programs have indeed been implemented and evaluated for more than 15 years in Latin America. Most of these programs included high-quality monitoring systems, as well as rigorous evaluation schemes, often as part of a research component supported by academic institutions, a strong support from governments, and appropriate funding. The positive and consistent impact of these CCT programs on poverty reduction has been well documented, which in part may explain their popularity and adoption by a number of countries in the region and beyond [START_REF] Arnold | Cash transfersliterature review[END_REF][START_REF] Fiszbein | Conditional cash transfers. Reducing present and future poverty[END_REF][START_REF] Handa | The experience of conditional cash transfers in Latin America and the Caribbean[END_REF]. The evidence on the impact of CCTs on food security and food consumption is also strong. A number of CT programs have been shown to increase household food expenditure and food consumption. In Colombia for example, household food consumption increased both in terms of quantity (energy intakes) and quality (intakes of nutrientrich foods such as fruits, vegetables, meat and dairy products) [START_REF] Attanasio | The impact of a conditional cash transfer programme on consumption in Colombia[END_REF]. Positive impacts on food consumption were also observed in Honduras and Mexico City [START_REF] Attanasio | Mexico's conditional cash transfer programme[END_REF][START_REF] Hoddinott | The impact of PROGRESA on food consumption[END_REF][START_REF] Wiesmann | The impact of a conditional cash transfer program on food consumption: The Honduras family allowance program (PRAF)[END_REF]). In the latter, as well as in Nicaragua, specific impacts on the consumption of fruits and vegetables and on dietary diversity have been shown [START_REF] Leroy | Cash and in-kind transfers in poor rural communities in Mexico increase household fruit, vegetable, and micronutrient consumption but also lead to excess energy consumption[END_REF][START_REF] Maluccio | Impact evaluation of a conditional cash transfer program: The Nicaraguan red de Proteccion social[END_REF].

Social transfer strategies that are successful in Latin America may not be easily transferable to sub-Saharan Africa, where constraints and development issues are different. In Africa, social protection programs, including food distribution, have historically been and remain mainly based on external assistance. The lack of capacity and resources to reach large numbers of poor and food insecure people, the restricted fiscal space, or the lack of political will are often given as explanations for reluctance of governments to adopt and scale-up such programs [START_REF] Adato | Linking safety nets, social protection, and poverty reduction-directions for Africa. Assuring Food and Nutrition Security in Africa 2020: Prioritizing actions, strengthening actors, and facilitating partnerships[END_REF][START_REF] Ellis | Social protection in Africa: Where we are, and how did we get there?[END_REF]. Two main types of social protection programs nevertheless exist on the continent. On the one hand, there are some true national social protection policies, usually in the form of pension schemes allocated by governments to specific groups (orphans, elderly, etc.) [START_REF] Devereux | Social pensions in southern Africa in the twentieth century[END_REF],who are not necessarily the most in need [START_REF] Adato | Exploring poverty traps and social exclusion in South Africa using qualitative and quantitative data[END_REF][START_REF] Devereux | Making cash count: Lessons from cash transfer schemes in east and southern Africa for supporting the most vulnerable children and households[END_REF]. These programs are mainly encountered in Southern Africa (South Africa, Namibia, Lesotho, Botswana and Swaziland) and have rapidly expanded to Eastern Africa (Kenya, Mozambique, Malawi, Uganda, Rwanda), with one exception in West Africa (Ghana). On the other hand, pilot projects, rather than national policies, are increasingly implemented throughout the continent. These projects are diverse in design and implementation, but share common features, at least in West Africa: they target the most vulnerable population groups, and are relatively small in scale, limited in time and supported by external donors. So far none of these pilot projects has become a government-owned social protection program with a national coverage, despite some attempts in Malawi and Ghana. A few large social transfer programs however exist in Eastern and southern Africa, and even one in Senegal, West Africa. These programs mostly aim at reducing poverty and/or improving food security or school attendance [START_REF] Kakwani | Conditional cash transfers in African countries[END_REF]. In 2008 the Transfer Project was initiated by UNICEF Innocenti, the FAO, the University of North Carolina and several national governments, with the objectives of sharing lessons learned, experiences and expertise from CT program evaluation in Africa. The unconditional CT programs included in this project benefited from rigorous evaluations, which concluded towards an improvement in household's food security either by the use of income for food or by the purchase of productive assets (livestock, agricultural equipment, fertilizers) [START_REF] Davis | From evidence to action: The story of cash transfers and impact evaluation in sub Saharan Africa[END_REF].

Despite these promising findings, the diversity of CT designs and modalities make it difficult to take stock from existing impact data. In particular, most of the evaluated programs were CT programs, while programs that distributed food or food vouchers are fewer and insufficiently evaluated. The International Food Policy Research Institute (IFPRI), on the basis of a 4-country study funded by the World Food Program (WFP), recently analysed the relative and absolute effectiveness of food versus cash versus voucher distributions in improving household food security and other measures of well-being. The findings indicated that the relative effectiveness of different modalities heavily depends on contextual factors such as the severity of food insecurity and the functioning of markets for grains and other foods [START_REF] Hoddinott | Enhancing WFP's capacity and experience to design, implement, monitor, and evaluate vouchers and cash transfer programmes: Study summary[END_REF]. In Democratic Republic of Congo, a randomized trial comparing cash versus vouchers found that both types of intervention had similar effects on total food expenditure [START_REF] Aker | Cash or coupons? Testing the impacts of cash versus vouchers in the Democratic Republic of Congo[END_REF].

The 2007-2008 food price crisis deeply affected Senegal, like many other countries in Sub-Sahara Africa. Poor households living in cities were particularly affected since their food supply depended entirely on markets. Between November 2007 and November 2008, a 39% increase in prices of unprocessed cereals was observed in the country, while the price of imported rice almost doubled (Agence Nationale de Statistique et de Démographie 2009). As the food vulnerability situation of urban households remained poorly known, the World Food Program (WFP) and collaborates carried out a food security survey in the three largest cities of the country (Pikine, Kaolack and Ziguinchor). The results revealed high rates of household's food insecurity and the use of negative coping strategies such as decreasing the number of meals consumed per day and/or quantities consumed, reducing the quality of meals, and even begging. Food expenditure also accounted for a large share of the household's budget in all three cities (56 to 59%). In such a context, and because markets were functional, the WFP and the government of Senegal identified a cash-based intervention as the most appropriate to alleviate the effect of the food crisis on household's food security. In 2010-2011WFP implemented a food voucher distribution for 6 months in poor households living in Dakar-Pikine (a vulnerable district of Dakar, the capital city) and in the smaller city of Ziguinchor, South Senegal. This paper presents the results of the impact evaluation of this program on household's food security and dietary diversity, using a quasiexperimental design.

The food voucher distribution program

The overall objective of the program was to compensate the decline in household's purchasing power caused by high food prices and reduced employment opportunities, in urban contexts where food products were however available. Dakar-Pikine was selected as the pilot area, where the program started in July 2010 and targeted 8806 vulnerable households. Lessons drawn from this first experience were used to implement a similar program in Ziguinchor in April 2011, which targeted 7137 vulnerable households. Quotas of households to be served was determined by financial constraints. Households were selected based on a participatory process in both cities: a local committee was in charge of providing a list of the most vulnerable districts, and of the most vulnerable households within each district, using pre-defined criteria presented in Table 1. In Ziguinchor, all districts were considered vulnerable and were, therefore, targeted by the program. In both cities the lists of households provided by local committees were checked by WFP staff using proxy-means test (PMT) questionnaires administered during home visits.

Depending on their PMT result, households were either maintained into or excluded from the program.

The program consisted in distributing paper vouchers worth 3000 FCFA (~US$5) to beneficiary households on a monthly basis during 6 months. One voucher per person was granted, with a maximum of 6 vouchers per household (i.e. a maximum of 18,000 FCFA per month and per household). Households redeemed the vouchers for pre-defined food products (rice, maize, sorghum, cooking oil and sugar) in shops that had signed a contract with WFP. In Dakar-Pikine only 6 distributions were organized within a 10-month period (July 2010-May 2011), due to logistical difficulties; in Ziguinchor 7 distributions were organized within a 7-month period (April-October 2011).

3 Material and methods for impact evaluation

Study design

The evaluation was based on a quasi-experimental design. This consisted in a non-randomized controlled longitudinal study where households who benefited the program were compared to households who did not, before ("baseline") and after ("endline") the program. Sampling procedures were slightly different between cities (Fig. 1). In Dakar-Pikine, a two-stage cluster sampling was used to randomly select targeted districts and beneficiary households from the lists of beneficiary districts and households provided by the program: 42 targeted districts were randomly selected proportionally to the number of inhabitants per district; 12 beneficiary households per district were then randomly selected from the lists. A total of 504 households were selected to participate in the study. A slightly different process was used to select non beneficiary households: 42 non-targeted districts were also randomly selected proportionally to the number of inhabitants per district; there was however no available list of vulnerable households living in these districts. A local committee was constituted to produce this list, using WFP's criteria to target beneficiary households (Table 1). Post-identification checking was however not performed by WFP. A total of 1008 vulnerable non-beneficiary households were identified using this process. A propensity score was calculated for each of these households using a logistic model predicting the probability of being a beneficiary, using a large set of demographic and socioeconomic variables [START_REF] Fortin | A propensity score to match exposed and unexposed groups when evaluating a food voucher programme[END_REF]. The 504 households who had the highest propensity scores were selected in the study in order to ensure the best comparability with the beneficiary households at baseline. The final sample size in Dakar-Pikine allowed detecting a 0.4 point difference in household dietary diversity score (HDDS) between groups, with a 5% level of significance and a 90% study power, and considering a 25% lost to follow-up between baseline and Impact of a food voucher program in alleviating household food insecurity in two cities in Senegal during a food price crisis endline and a cluster effect of 1.5. In Ziguinchor, a simple random sampling, stratified by district, was used to sample a total of 498 households from the list of beneficiaries. This sample size allowed detecting a 0.3 difference in HDDS between groups. The same number of non-beneficiary households was selected in each district, from a supplementary list of vulnerable households provided by the local committee.

Data collection

The baseline survey in Dakar-Pikine was conducted in May 2010, before the first vouchers were distributed, and the endline survey was conducted just after the last voucher distribution, in May 2011. In Ziguinchor the baseline and endline surveys were conducted in December 2010 and 2011, respectively. Unexpected delays in intervention implementation caused a 3-month gap between the baseline and the first distribution in Ziguinchor. Data were collected by trained enumerators through face-to-face interviews during home visits, using pre-tested questionnaires and Personal Digital Assistant. The respondent was preferably the household head or the first spouse otherwise. The questionnaire included detailed information on household's sociodemographic characteristics, education, housing quality, assets, food consumption, food insecurity, coping strategies, food and non-food expenditures, debts and savings, as well as on the mothers' and children's characteristics and children food consumption. The endline questionnaire also included a section on household's experience and satisfaction with the voucher distribution.

Outcome indicators

Household dietary diversity score (HDDS)

The HDDS is an established proxy measure of household food access [START_REF] Swindale | Household dietary diversity score (HDDS) for measurement of household food access: Indicator guide[END_REF], which is the ability of a household to acquire food in sufficient quality and quantity to meet all household members' nutritional requirements for productive lives. The score is calculated by summing up the number of food groups consumed at home by any of the household members in the previous 24 h from 12 food groups (cereals, roots and tubers, vegetables, fruits, meat and poultry, eggs, fish and seafood, pulses/legumes/nuts, milk and milk products, oils and fats, sugar and honey, and condiments). The HDDS theoretically varies from 0 to 12.

Household food insecurity access scale (HFIAS)

The HFIAS is an established measure of food security that has been developed by Food And Nutrition Technical Assistance (FANTA) project [START_REF] Coates | Household food insecurity access scale (HFIAS) for measurement of food access: Indicator guide[END_REF]. It consists of 9 specific statements about the accessibility of foods for the household's members as well as household's concern about food. The respondent, preferentially the head of household or the woman in charge of food provisioning/cooking, is asked whether each statement happened at all in the past 30 days (yes or no) and, if yes, how often it occurs over the period. A score is allocated for each question as follows: zero if the statement never occurred, 1 point if it occurred 1 or 2 times (rarely), 2 points if it occurred 3-10 times (sometimes), and 3 points if it occurred >10 times (often). These points are summed up to compute the HFIAS, ranging from 0 (food security) to 27 (maximum food insecurity). In this study the HFIAS was divided into 2 categories to determine severe food insecurity versus other categories, as defined in the FANTA guide [START_REF] Coates | Household food insecurity access scale (HFIAS) for measurement of food access: Indicator guide[END_REF]).

Coping strategies index (CSI)

Households were asked what kind of strategy (over a list of 5 pre-determined strategies) they used in case they did not have enough food or enough money to buy food in the past 7 days [START_REF] Maxwell | The coping strategies index: Field method manual[END_REF]. The frequency of use of these strategies was taken into account, as well as standard weights allocated to each strategy depending on its severity. The index may vary from 0 (no strategy is used) to 28 (maximum food insecurity).

Household food expenditure

Food expenditure were recorded using a recall of expenses on the previous day, week, or month for items bought daily, weekly, or monthly, respectively. All expenses were then converted into daily food expenditure, summed up, and divided by the total number of adult-equivalents (AE) in the household, calculated based on individual energy requirements [START_REF] Fao | Human energy requirements -report of a joint FAO/WHO/UNU expert consultation[END_REF]. Food expenditures were log transformed and geometric means are presented in this paper.

Demographics

Basic demographic data such as age, gender, and size of household were collected. A youth ratio (number of household members <15 y of age/number of persons > = 15 y) and an economic dependency ratio (number of people not contributing to household expenditure/number of people contributing) were also computed.

Data management and statistical analysis

Data cleaning, management and analysis were performed using SAS 9.3 (SAS Institute Inc., Cary, NC, USA). Comparability of households' characteristics at baseline between intervention and control groups was checked first. Results are presented by group, in means ± SD for quantitative variables and in frequency ± SE for qualitative variables. Statistical models used for the comparisons (beneficiary vs. non-beneficiary) were linear regression for quantitative continuous variables and logistic regression for qualitative binary variables. To assess the impact of the interventions a difference-in-differences approach was used [START_REF] Lance | How do we know if a program made a difference? A guide to statistical methods for program impact evaluation[END_REF]. Mixed models were computed with each of the outcome indicators as dependent variable. Fixed effects were the status of group (beneficiary/non beneficiary), the survey (baseline/endline), their interaction (status of Group X Survey), as well as potential confounding factors. Random effects were the cluster design effect (only in Dakar-Pikine) and the repetition of measurements in the same households. In all models, the interaction term Group X Survey enabled detecting a change over time between the beneficiary and nonbeneficiary groups. Potential confounding factors were variables for which level changed differently in the two groups between baseline and endline. These factor were identified in separate regression models including an interaction term "status of Group X Survey"; variables were retained as confounding factors if this interaction term was statistically significant at p < 0.15 to account for the lower power of interaction tests compared with main effects [START_REF] Brookes | Subgroup analyses in randomized trials: Risks of subgroup-specific analyses; power and sample size for the interaction test[END_REF]). The analysis was performed on an "intent-to-treat" basis with a level of significance fixed at 5%. In Dakar-Pikine, observations were weighted according to the sampling scheme.

Ethics

The protocol of the study was submitted for ethical clearance from the National Ethical Committee of Research, Ministry of Health, Dakar, Senegal on 2010, April 19th (Authorization No SEN12/10). Informed consent was obtained from all individual participants included in the study.

Results

Comparison of beneficiary and non-beneficiary households at baseline

Beneficiary and non-beneficiary households had similar sociodemographic characteristics at baseline, in both cities (Table 2). In Ziguinchor, the only differences detected between groups were the proportion of household heads owning their house (58.5% for non-beneficiaries versus 48.4% for beneficiaries, p = 0.02) and their dependency ratio (p = 0.007).

Changes in basic characteristics of households between baseline and endline

Most sociodemographic characteristics of households did not vary between baseline and endline, or varied in the same way between beneficiary and non-beneficiary groups. In Dakar-Pikine however, the average household size increased from 11.7 persons at baseline to 12.9 people at endline among beneficiaries, whilst it increased from 10.9 to 12.5 people among non-beneficiaries (p value for interaction = 0.02). Household size was therefore considered as a potential confounding factor and added as covariate in all analyses regarding Dakar-Pikine samples.

Impact of a food voucher program in alleviating household food insecurity in two cities in Senegal during a food price crisis Author's personal copy 4.3 Impact measurement

Household dietary diversity

The mean HDDS decreased between baseline and endline in Dakar-Pikine, whether the households received the vouchers or not (-0.7 and -0.5 points respectively, p value for interaction = 0.2) (Fig. 2). This was due to a decrease in the frequency of consumption of fish and roots/tubers, while a significant increase in frequency of consumption of condiments was also observed in this city (Fig. 3a). In Ziguinchor, a decrease in HDDS between the two rounds was observed in all households; however the decrease was stronger in nonbeneficiary households than in beneficiary households (-0.6 points versus -0.3 points, p value for interaction = 0.02). A decrease in the frequency of consumption of fats, vegetables a Model used: linear regression for quantitative continuous variables (Proc surveyreg) and logistic regression for qualitative binary variables (Proc surveylogistic), with the districts considered as clusters b HH = head of household and legumes was indeed observed in non-beneficiary households, but not in beneficiary households (Fig. 3b).

Household food insecurity

In Ziguinchor, the proportion of severely food insecure households significantly decreased in the group that received vouchers between baseline and endline (from 83.9% to 64.6%), while it remained almost the same in the nonbeneficiary group (p value for interaction = 0.0003) (Table 3). The risk of being severely food insecure after the intervention was 1.6 times higher in non-beneficiary households compared to beneficiary households, while the corresponding figure at baseline was 0.7. In Dakar-Pikine, the proportion of severely food insecure households remained stable in both groups (p value for interaction = 0.17).

Coping strategies

In Ziguinchor, the mean CSI did not vary between the two rounds of survey in beneficiary households (approximately 18 at baseline and endline), while it increased from 16.9 to 22.5 in non-beneficiary households (p value for interaction <0.0001), indicating a higher food insecurity at endline in these households (Table 3). The mean CSI in Dakar-Pikine decreased between baseline and endline survey, whether households received the vouchers or not (p value for interaction = 0.41).

Household food expenditure

Monthly food expenditures per adult-equivalent decreased between baseline and endline in all households in both cities (pvalues for interaction = 0.39 in Dakar-Pikine, 0.43 in Ziguinchor) (Fig. 4).

Use of vouchers

At endline a questionnaire was administered to beneficiary households in both cities in order to investigate how the vouchers were used. Results refer to a subsample of 427 households in Ziguinchor and 352 households in Dakar-Pikine. In both cities in exchange for the vouchers, beneficiary households mainly chose rice (97.2 and 91.5% in Ziguinchor and Dakar-Pikine, respectively), cooking oil (87.7 and 78.1%) and sugar (64.6 and 30.6%). Sorghum was chosen by less than 11% of households in both cities and maize was barely chosen (<1% in both cities). In Ziguinchor, only 5.6% of households (n = 24) wished that they could exchange vouchers with other items than those authorized within the program, while they were 38% (n = 134) in Dakar-Pikine. The most cited other items that households would have liked to get were butter and chocolate.

Twenty-three percent of households in Dakar-Pikine, and 29% of households in Ziguinchor managed to make savings thanks to the intervention; approximately half of them (58% in Dakar-Pikine and 46% in Ziguinchor) "invested" these savings, mainly in reimbursing debts or in health expenditure. Finally, they were 68.9% households in Dakar-Pikine and 46.2% in Ziguinchor who shared vouchers with family, neighbours or friends (by giving foods exchanged for the vouchers or by inviting people to share their meals).

Discussion

The overall state of food security worsened between baseline and endline in both the studied cities and in both nonbeneficiary and beneficiary households. This is particularly true for the HDDS and monthly food expenditure, which decreased in all households between the two surveys. In Ziguinchor however, households who received vouchers were less affected by this general deterioration than households who did not. Using the HFIAS indicator, the risk of being severely food insecure after the intervention was 1.6 times higher in non-beneficiary households compared to beneficiary households. These findings suggest a protective effect of the intervention on household's food security in this city. In Dakar-Pikine, the intervention had no visible protective effects on any of the food security indicators analysed.

Most unconditional CT programs evaluated in sub-Saharan Africa have resulted in improved household's food security, via the purchase of food or the purchase of productive assets (e.g. livestock, agricultural equipment, fertilisers). This was the case in Malawi [START_REF] Brugh | Impacts of the Malawi social cash transfer program on household food and nutrition security[END_REF][START_REF] Miller | The impact of the social cash transfer scheme on food security in Malawi[END_REF], Ethiopia [START_REF] Berhane | The impact of Ethiopia's productive safety nets and household asset building programme: 2006-2010[END_REF][START_REF] Debela | Does Ethiopia's productive safety net program improve child nutrition?[END_REF], Zambia [START_REF] Schuering | Experiences with social cash transfers as a tool to mitigate the impact of AIDS in rural families in Zambia[END_REF][START_REF] Seidenfeld | Results of the three year impact evaluation of Zambia's cash transfer program in Monze District Washington[END_REF] and Zimbabwe [START_REF] Bhalla | The effect of cash transfers and household vulnerability on food security in Zimbabwe[END_REF] for example. CT programs that failed to demonstrate an impact on food security or related indicators usually conjured up irregular, unpredictable payments or low transfer value to explain this lack of impact [START_REF] Evans | Communitybased conditional cash transfers in Tanzania: results from a randomized trial[END_REF]FAO 2014a, b) [START_REF] Houngbe | Effects of multiannual, seasonal unconditional cash transfers on food security and dietary diversity in rural Burkina Faso: The moderate acute malnutrition out (MAM'Out) cluster-randomized controlled trial[END_REF][START_REF] Tiwari | Impact of cash transfer programs on food security and nutrition in sub-Saharan Africa: A cross-country analysis[END_REF]. The literature on food vouchers and food transfers in sub-Saharan Africa is far less abundant, out of emergency contexts. In the Democratic Republic of Congo, Aker demonstrated that food voucher distributions to households increased the number of meals per day and decreased the likelihood of suffering from food insecurity [START_REF] Aker | Cash or coupons? Testing the impacts of cash versus vouchers in the Democratic Republic of Congo[END_REF]. In Niger and Uganda, this type of intervention resulted in an increase in household's food security as measured by the Food Consumption Score, the Dietary Diversity Index or by the use of fewer coping strategies [START_REF] Hoddinott | Enhancing WFP's capacity and experience to design, implement, monitor, and evaluate vouchers and cash transfer programmes: Study summary[END_REF]. In rural Malawi, Gelli et al. reported a protective effect of food transfers during the lean season on household food security [START_REF] Gelli | Lean-season food transfers affect Children's diets and household food security: Evidence from a quasi-experiment in Malawi[END_REF]).

Program's failure or suboptimal success may be either due to a failure in conception or a failure in implementation, or Impact of a food voucher program in alleviating household food insecurity in two cities in Senegal during a food price crisis both. The food voucher program we evaluated was implemented in a crisis context, where food prices increased sharply. Yet the value of food vouchers was calculated long before the intervention started, and never was readjusted over the course of the program. Setting the suitable transfer amount that will achieve the stated program's objective is essential for any CT program [START_REF] Davis | How much do programmes pay? Transfer size in selected national cash transfer programmes in sub-Saharan Africa[END_REF]. The overall decline in food security indicators we observed may be partly explained by the inadequacy of the amount received to face this crisis, especially in Dakar-Pikine where the cost of living was high. In the meantime, households also had to face the rise in prices related to non-food products (rent, transportation, bills, etc.) which probably further contributed to the reduction in their monthly food expenditure. The fact that households could exchange the vouchers against a limited set of foods (rice, maize, sorghum, cooking oil and sugar) did not permit an increase in household's dietary diversity. Recipients primarily used the vouchers to ensure immediate consumption needs such as rice and oil which are key ingredients of local family meals. The hypothesis of the program was to provide staple foods to households, so that they could save money and purchase nutritious foods such as meat, fish or fruits and vegetables. Clearly this mechanism was not observed, probably due to the generalized increase of food and non-food prices.

Other food voucher or food basket programs offering more diverse set of items (including fresh foods or fortified foods) led to improved household or individual dietary diversity [START_REF] Acf | Meta-evaluation of ACF fresh of food voucher programmes[END_REF][START_REF] Aker | Cash or coupons? Testing the impacts of cash versus vouchers in the Democratic Republic of Congo[END_REF][START_REF] Hidrobo | Cash, food, or vouchers? Evidence from a randomized experiment in northern Ecuador[END_REF][START_REF] Ramírez-Luzuriaga | A food transfer program without a formal education component modifies complementary feeding practices in poor rural Mexican communities[END_REF]. In rural Mexico, a food basket distribution significantly increased the prevalence of 6-23 months old children reaching minimum dietary diversity. In Ecuador, a food voucher transfer was combined with nutrition sensitization -through monthly meetings and flyers purposely developed and posted at distribution sites -aimed at influencing behaviour change and increasing knowledge especially with regard to dietary diversity. One can wonder whether integrating an educational component into the intervention (for example recommendations on food items to buy along with the vouchers), or merely offering more diversity of food items would have helped the program achieve its objectives. Nutrient-rich foods such as dried meat, canned sardines or tuna, or milk could have been considered for inclusion into the list of authorized foods to exchange for vouchers without increasing logistical constraints. Moreover, because the program was very short in duration, a non-negligible share of households did not use the vouchers to improve their diet, but rather for other purposes such as reimbursing debts or covering medical expenses. While there is no doubt that the most needy must be accurately identified for effective poverty and food insecurity alleviation, the way the poorest can be reached is a long-lasting concern. Proxy-mean testing, community-based and geographical targeting are different approaches which have been proved more or less efficient depending on the contexts [START_REF] Fortin | Targeting vulnerable households in urban Burkina Faso: Effectiveness of geographical criteria but not of proxy-means testing[END_REF][START_REF] Robertson | Involving communities in the targeting of cash transfer programs for vulnerable children: Opportunities and challenges[END_REF][START_REF] Stoeffler | Reaching the poor: Cash transfer program targeting in Cameroon[END_REF]). In the present study, a community-based targeting coupled with proxy-mean test checking was used to select recipients. Indicators of household well-being (HH dietary diversity, food expenditure and food security) were all extremely low in Ziguinchor, suggesting that very poor households were truly targeted. Despite the well-known poverty of Dakar-Pikine, overall indicator values were not so low (except food insecurity), suggesting that the targeted population could better cope one way or another. Complementary analyses were carried out to further explore the impact of the intervention on the poorest households in Dakar-Pikine, using the lowest quintiles of a household wealth index (calculated by multiple correspondence analysis, based on the household assets, quality of housing and equipment, access to water, and number of people living in the same room at baseline). In the poorest quintiles (n = 172), a protective effect of the intervention on HH dietary diversity and food security was noted, similar to the one observed in Ziguinchor (p-values for interaction = 0.007 and 0.005 respectively, results not shown). While firm conclusions cannot be made from this subanalysis on a reduced sample of extremely poor households, the trend indicates that the cash-voucher transfer does a better job when reaching the very poor groups.

The intervention was first implemented in Dakar-Pikine, and programmatic difficulties were observed. This included irregularity and unpredictability in distributions (6 distributions in 10 months), which are factors often cited as major contributors to low impact of CT interventions [START_REF] Evans | Communitybased conditional cash transfers in Tanzania: results from a randomized trial[END_REF]FAO 2014a;[START_REF] Handa | Livelihood empowerment against poverty program impact evaluation[END_REF]). Significant program deviation was also observed in Dakar-Pikine. At endline, 56 beneficiary households did not receive vouchers at each distribution, representing 14.2% of the total sample. Among them, 44 households had not received anything. Inversely, 17 households who were supposed to be non-beneficiary actually received some coupons, representing 3.7% of the nonbeneficiary households. This may have contributed to the dilution of effect in this city. The experience acquired in Dakar-Pikine was certainly useful to implement the same program in Ziguinchor since none of these problems occurred there. Moreover a larger proportion of households did share the vouchers they received with friends and family in Dakar-Pikine than in Ziguinchor, probably due to higher social pressure, and to lower coverage of the program (estimated at 8.8% in Dakar-Pikine and 28.5% in Ziguinchor).

The evaluation study had some limitations. In Dakar-Pikine the fact that the program targeted the most vulnerable households made the selection of non-beneficiary households challenging. The issue was to find households who were as poor as the beneficiary households, even though they had not been targeted by the program (hence not considered as the poorest). It was decided to select non beneficiary households from neighbouring districts that were not targeted by the program for ethical reasons, to avoid risks of "contamination" between groups, and because it was deemed that the poorest households would have been selected to receive the vouchers. In these districts, the non-beneficiary households were selected using a process similar to the one used by the program to select the beneficiary households (list of the most vulnerable households provided by a local committee and based on predefined criteria), except the post-identification checking that could not take place because of logistical and financial constraints. The final strategy consisted of doubling the sample size of non-beneficiary households so that half of themthe most comparable to beneficiary households using propensity matchingcould be selected for impact evaluation. This strategy appeared to be efficient except for the monthly food expenditure, which was higher in non-beneficiary household compared to beneficiary households at baseline. This was nevertheless the best strategy we could use in the absence of random assignment to experimental and control groups. In Ziguinchor the sampling procedure was more straightforward because all districts were considered as poor and were therefore targeted by the program. Non-beneficiary households were directly and randomly selected from a complementary list of poor households provided by the local committee. Larger sample size may have allowed to detect more significant changes between groups.

The indicators for household food security and well-being worsened between baseline and endline in both cities and in both groups of households, due to the economic and price crisis that occurred during the intervention. The pilot distribution of food vouchers alleviated some effects of the crisis in Ziguinchor, but not in Dakar-Pikine. In the latter city, more regular transfers, adjustment of transfer size over time and/or targeting the extreme poor may have helped the program meet its objectives.

Fig. 1

 1 Fig. 1 Sampling procedure for beneficiary and non-beneficiary households in Dakar-Pikine and Ziguinchor

Fig. 2

 2 Fig. 2 Change in the mean household dietary diversity score in beneficiary and nonbeneficiary households

Fig. 3

 3 Fig. 3 Change in the frequency of consumption of HDDS items in beneficiary and non-beneficiary households between baseline and endline in (a) Dakar-Pikine and (b) Ziguinchor

Fig. 4

 4 Fig. 4 Change in monthly food expenditure between baseline and endline (in FCFA per adultequivalent)

Table 1

 1 List of criteria used for the targeting of the intervention in

	Dakar-Pikine
	Criteria for selection of the most vulnerable districts
	Flooding
	Access to safe drinking water
	Sanitation/waste management
	Population density
	Health services
	Schooling services
	Housing scheme
	Access to electricity
	Criteria for selection of the most vulnerable households within districts
	Food consumption (based on the number of meals/day)
	Presence of children with malnutrition
	Presence of at least two children under 5
	Household member with handicap or chronic disease
	Presence of a pregnant women
	More than 2 persons older than 65 years
	Household surviving from begging
	Household whose house was flooded
	Household living in a hut

Table 2

 2 Comparison household's characteristics at baseline

		Dakar-Pikine		P	Ziguinchor		P
				value a			value a
		Beneficiary	Non beneficiary		Beneficiary	Non beneficiary
		N Mean (SE) or	N Mean (SE) or		N Mean (SE) or	N Mean (SE) or
		Frequency [95%	Frequency [95%		Frequency [95%	Frequency [95%
		CI]	CI]		CI]	CI]
	Age of HH b (year)	377 54.5 (0.9)	424 55.4 (0.7)	0.44	409 53.7 (0.6)	404 54.0 (0.7)	0.70
	Household size (No of	400 11.7 (0.4)	454 10.9 (0.3)	0.10	422 9.9 (0.2)	416 10.3 (0.3)	0.21
	persons)
	Youth ratio	399 0.74 (0.04)	453 0.70 (0.04)	0.51	422 0.67 (0.03)	415 0.65 (0.04)	0.81
	Dependency ratio	354 3.5 (0.2)	391 3.21 (0.14)	0.28	400 3.8 (0.2)	413 4.4 (0.2)	0.007
	Gender of HH	398	452	0.34	417	410	0.05
	Male	57.2 [49.9-64.6]	52.6 [4.6-58.7]		54.5 [49.6-59.3]	47.4 [42.5-52.3]
	Female	42.8 [35.4-50.1]	47.4 [41.3-53.4]		45.5 [40.7-50.7]	52.6 [47.7-57.5]
	Matrimonial status of HH	401	458	0.23	430	428	0.19
	Single	5.8 [1.1-10.6]	3.9 [1.7-6.1]		7.1 [4.7-9.5]	6.5 [4.1-8.8]
	1 spouse	46.3 [39.8-52.8]	43.7 [38.6-48.7]		48.8 [43.7-53.3]	46.1 [41.1-50.6]
	> 1 spouse	25.0 [20.5-29.5]	25.1 [20.7-29.5]		15.8 [12.2-19.3]	15.9 [12.4-19.3]
	Divorced	2.1 [0.6-3.7]	3.1 [1.6-4.5]		3.9 [2.0-5.7]	3.2 [1.5-4.9]
	Widow	20.7 [13.9-27.4]	24.2 [19.1-29.4]		24.5 [20.3-28.6]	28.4 [24.0-32.7]
	Education of HH	400	458	0.48	430	428	0.96
	None	58.7 [51.3-66.1]	55.5 [48.9-62.0]		57.9 [53.2-62.6]	57.0 [52.3-61.8]
	Coranic school	20.4 [15.6-25.3]	21.2 [17.1-25.3]		7.9 [5.3-10.4]	9.4 [6.6-12.1]
	Primary school	13.8 [8.9-18.7]	14.8 [11.0-18.6]		23.3 [19.3-27.3]	23.6 [19.5-27.6]
	Secondary school	6.5 [2.6-10.4]	7.9 [5.3-10.4]		10.5 [7.6-13.5]	9.1 [6.3-12.0]
	Higher school	0.5 [0.0-1.5]	0.7 [0.0-1.4]		0.4 [0.00-1.0]	0.9 [0.01-1.8]
	Profession of HH	400	458	0.93	430	428	0.33
	Employed	45.3 [37.3-53.3]	43.9 [38.8-49.0]		70.0 [65.6-74.4]	65.6 [61.0-70.1]
	Job seeker	5.4 [3.0-7.8]	7.0 [4.1-9.9]		6.0 [3.7-8.3]	5.8 [3.6-8.0]
	Housewife	32.2 [24.7-39.7]	34.1 [28.6-39.5]		17.3 [13.6-20.9]	22.5 [18.5-26.5]
	Retired	11.2 [6.9-15.5]	10.5 [7.5-13.4]		4.5 [2.6-6.5]	2.9 [1.3-4.5]
	Other	5.9 [1.6-10.2]	4.6 [2.4-6.7]		2.2 [0.4-2.6]	3.2 [1.5-4.9]
	Housing status	400	458	0.53	430	428	0.02
	Owner	65.1 [58.8-71.3]	60.9 [54.0-67.2]		48.4 [43.7-53.2]	58.5 |53.8-63.2]
	Co-owner	7.6 [3.9-11.3]	12.2 [7.6-16.9]		8.5 [5.8-11.2]	5.8 [3.6-8.0]
	Tenant	15.7 [11.6-19.9]	15.5 [10.9-20.1]		16.0 [12.4-19.5]	15.2 [11.8-18.7]
	Co-tenant	4.9 [1.9-7.8]	5.0 [2.2-7.8]		1.9 [0.6-3.3]	3.3 [1.6-5.0]
	Housed for free	6.7 [3.8-9.6]	6.3 [4.5-8.2]		25.1 [21.0-29.3]	17.2 [13.5-20.8]

Table 3

 3 Change in food insecurity indicators between baseline and endline P-value for the interaction term (Group x Survey) in mixed models with fixed effects: status of group (beneficiary/non beneficiary), survey (baseline/ endline), their interaction (status of group X survey), size of households (only in Dakar-Pikine) and random effects: cluster design effect (only in Dakar-Pikine) and the repetition of measurements in the same households

		Dakar-Pikine				p-	Ziguinchor		p-value a
						value a
		N Baseline		Endline			N Baseline		Endline
		Mean (SE) OR[95%	Mean (SE) OR[95%		Mean (SE) OR[95%	Mean (SE) OR[95%
		or	CI]	or	CI]		or	CI]	or	CI]
		Frequency		Frequency			Frequency		Frequency
		[95% CI]		[95% CI]			[95% CI]		[95% CI]
	Proportion of severely food insecure households (HFIAS)
	Beneficiary 401 80.7	0.9	80.2	0.7	0.17 428 83.9	0.7[0.5-0.9] 64.6	1.6[1.2-2.1] 0.0003
		[75.1-86-	[0.6--	[72.8-87-	[0.4--		[80.4-87-		[60.0-69-
		.3]	1.5]	.6]	1.1]		.4]		.1]
	Non	458 80.2		71.6			428 77.9		73.7
	benefi-	[72.8-87-		[64.1-79-			[73.9-81-		[69.5-77-
	ciary	.6]		.1]			.9]		.9]
	Coping Strategy Index
	Beneficiary 400 21.4 (0.9)		19.5 (0.9)		0.41 429 18.5 (0.6)		18.3 (0.6)	<0.0001
	Non	458 20.2 (0.9)		17.3 (0.9)			428 16.9 (0.6)		22.5 (0.6)
	benefi-
	ciary

a

Funding The research was funded by the World Food Program.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent Informed consent was obtained from all individual participants included in the study. Atsuvi Gamli is the W FP Regional Market Analyst at Regional Bureau of Dakar (RBD). His responsibilities include the analysis of all relevant regional and national food and nutrition security and market. He has more than 16 years of experience, having served as VAM Officer in WFP Chad country office, Head of VAM/M&E Unit in WFP Mauritania country office. He joint WFP Senegal in 2008 as consultant. Before joining WFP, he worked with several market research companies in Senegal and West Africa Region. He received a master's degree in social protection from the University of Grenoble (France), a master's degree in marketing and communication of ISM Dakar and master degree (engineer) in statistics from ENEA-Dakar.

Mathilde

Kokou Amouzou is currently project manager at FAO Haiti as food security and nutrition specialist. He supports the H a i t i a n g o v e r n m e n t i n strengthening the governance of food security and nutrition. Previously, he was head of V A M ( V u l n e r a b i l i t y Assessment and Mapping) unit at WFP Haiti where he was responsible for food security and market analyses and vulnerability targeting coordination. Before joining Haiti, he was Data Analyst at WFP Senegal. In all, he has over 10 years of experience in project management, monitoring and evaluation Since September 2016 he is also Director of the "Health and Societies" Scientific Department of the IRD. He has been working for long at the interface between scientific and operational institutions as an expert serving various Committees and Task Forces for international organizations. He was also a member of the Independent Expert Group for the Global Nutrition Report from 2014 to 2017 and is currently a member of the Technical Advisory Committee of the Compact2025 initiative.


Fabien Pierrel

Megan L Bestwick

Paul A Cobine

Oleh Khalimonchuk

Julia A Cricco

Dennis R Winge
email: dennis.winge@hsc.utah.edu

Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly

Keywords: cytochrome c oxidase, Shy1, Mss51, heme a, mitochondria, copper 2

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Δ cells, but these subunits fail to accumulate. The respiratory defect in coa1Δ cells is suppressed by high copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Δ and shy1Δ cells, is enhanced when Mss51 and Cox10 are co-expressed. Shy1 has been implicated in formation of the heme a 3 -Cu B site in Cox1. The interaction between Coa1 and nascent Cox1 and the physical and genetic interactions between Coa1 and Mss51, Shy1 and Cox14 suggest that Coa1 coordinates the transition of nascent Cox1 from the Mss51:Cox14 complex to the heme a cofactor insertion involving Shy1. coa1Δ cells also display a mitochondrial copper defect suggesting that Coa1 may have a direct link to copper metallation of CcO.

INTRODUCTION

Cytochrome c oxidase (CcO) is the terminal enzyme of the energy transducing respiratory chain in mitochondria of eukaryotes, coupling the reduction of molecular oxygen to proton translocation across the inner membrane (IM) which drives ATP synthesis. Eukaryotic CcO consists of 12-13 subunits, with three mitochondrial encoded subunits (Cox1-Cox3) forming the core enzyme. The remaining subunits are encoded by the nuclear genome and consist of small polypeptides that surround the catalytic core [START_REF] Tsukihara | Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8A[END_REF]. The core enzyme contains three copper atoms and two heme a, a cofactor specific to CcO [START_REF] Tsukihara | Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8A[END_REF]. The enzyme is embedded within the IM with a portion of the molecule protruding into the intermembrane space (IMS) and a separate portion extending into the matrix compartment.

Assembly of CcO requires a myriad of steps including the assembly of subunits translated on cytoplasmic and mitochondrial ribosomes, modification of protoheme to heme a and the subsequent insertion of this moiety, along with that of copper, zinc and magnesium ions, into the nascent enzyme complex [START_REF] Carr | Assembly of cytochrome c oxidase within the mitochondrion[END_REF][START_REF] Khalimonchuk | Biogenesis of cytochrome c oxidase[END_REF]. Two separate copper centers Cu A and Cu B are functionally important in both Cox2 and Cox1, respectively. The assembly process of CcO requires over 30 accessory proteins (Barrientos et al., 2002a;[START_REF] Tzagoloff | PET Genes of Saccharomyces cerevisiae[END_REF] and appears to follow a sequential order of subassembly intermediates in mammalian tissues [START_REF] Nijtmans | Assembly of cytochrome c oxidase in cultured human cells[END_REF][START_REF] Williams | Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1 or SURF1[END_REF].

Translation of mitochondrial-encoded subunits occurs within the matrix on ribosomes associated with both the inner boundary membrane and cristae membrane [START_REF] Vogel | Dynamic subcompartmentalization of the mitochondrial inner membrane[END_REF].

Transcripts for COX1-COX3 are recruited to the IM in yeast by a series of IM-associated translational activators [START_REF] Sanchirico | Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs[END_REF]. These activators bind sequences in the 5' untranslated regions of the mRNA and promote co-translational membrane insertion [START_REF] Naithani | Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae[END_REF]. Translation of the COX1 transcript is activated by Pet309 [START_REF] Manthey | The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae[END_REF], the COX2 transcript by Pet111 [START_REF] Green-Willms | Pet111p, an inner membrane-bound translational activator that limits expression of the Saccharomyces cerevisiae mitochondrial gene COX2[END_REF], and the COX3 transcript by Pet54, Pet122 and Pet494 (McMullin and Fox, 1993). In addition to Pet309, Mss51 is required for COX1 translational initiation and has an additional role in translational elongation [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF][START_REF] Perez-Martinez | Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p[END_REF][START_REF] Siep | Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae[END_REF]. Mss51 also binds Cox1 nascent chains and collaborates with Cox14 in a post-translational step in CcO assembly [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. Cox1-Cox3 are inserted into the IM by translocases, one of which is Oxa1 [START_REF] He | Membrane translocation of mitochondrially coded Cox2p. Distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p[END_REF][START_REF] Hell | Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA[END_REF].

Heme a biogenesis involves farnesylation and oxidation of protoheme catalyzed by Cox10 and Cox15, respectively. Although eukaryotic heme a insertion is not well understood, heme a is likely inserted in Cox1 prior to its association with other subunits [START_REF] Williams | Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1 or SURF1[END_REF]. In the absence of Cox2, Cox1 accumulates in Rhodobacter sphaeroides with a single heme a moiety in the heme a site leaving the a 3 site unpopulated [START_REF] Bratton | Identification of the structural subunits required for formation of the metal centers in subunit I of cytochrome c oxidase of Rhodobacter sphaeroides[END_REF]. Heme a 3 formation likely occurs concurrent with Cox11-mediated Cu B site formation in Cox1. Shy1 appears to be important for heme a 3 insertion or stabilization of the intermediate prior to Cox2 binding because R. sphaeroides lacking SHY1 have a CcO assembly defect in heme a 3 and Cu B [START_REF] Smith | Assembly of cytochrome c oxidase in the absence of the assembly protein Surf1p leads to loss of the active site heme[END_REF]. Shy1 is not absolutely required for CcO assembly in yeast, since shy1Δ cells retain 10-15% residual CcO activity. Mutations in SURF1, the human ortholog of SHY1, cause a CcO deficiency resulting in Leigh syndrome [START_REF] Zhu | SURF1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome[END_REF].

Copper insertion is required in the maturation of both Cox1 and Cox2. A number of accessory factors are important in the copper metallation of CcO within the IMS. The source of copper for the formation of the Cu A and Cu B cofactor sites in CcO derives from a copper pool within the mitochondrial matrix. This pool of copper is present in a ligand complex (CuL) that is conserved in eukaryotic cells (Cobine et al., 2006a). Targeting of heterologous copper-binding proteins to the matrix attenuates this CuL complex resulting in an impairment of CcO assembly. This predicts the presence of mitochondrial IM proteins for the transport of the CuL complex (Cobine et al., 2006b).

A group of mitochondrial proteins of unknown function were designated as the Found in Mitochondrial Proteome (FMP) family in a yeast proteomic study [START_REF] Sickmann | The proteome of Saccharomyces cerevisiae mitochondria[END_REF]. In an attempt to identify proteins involved in the translocation of the CuL complex to mitochondria, we screened a collection of FMP gene deletion strains for those exhibiting respiratory defects and abnormal mitochondrial copper concentrations. In the present study, we identify one FMP protein (Yil157c/Fmp35) that when deleted from cells results in low mitochondrial copper levels and attenuated CcO activity. This IM protein, renamed Coa1 for Cytochrome Oxidase Assembly 1, is shown presently to be important for Cox1 assembly. Deletion of COA1 or SHY1 leads to very similar phenotypes that can be suppressed by MSS51 and COX10 arguing that Coa1 and Shy1 function at a closely related step in Cox1 assembly. Genetic and physical interaction of Coa1 with Mss51, Cox14 and Shy1 suggest that Coa1 is important for coordinating Cox1 translation and cofactor assembly in this subunit.

RESULTS

coa1Δ cells are respiratory deficient and have low level of mitochondrial copper

Mitochondria isolated from 54 FMP deletion strains were screened by ICP/OES analysis for abnormal mitochondrial copper levels. One strain, coa1Δ (yil157cΔ), showed reduced mitochondrial copper (Fig. 1A). Copper levels were 43% of WT in gradient purified mitochondria, yet total cellular copper was unaffected. This defect was copper specific as zinc, iron and manganese levels were not altered in coa1Δ cells. Mitochondria isolated from the deletion cells cultured in 0.5 mM CuSO 4 contained the same elevated levels of copper as WT cells (Fig. 1A). As copper is required for CcO function, we quantified oxygen consumption of coa1Δ cells. This deletion strain consumed less oxygen than the WT and the addition of 1 mM CuSO 4 to the growth medium partially enhanced respiration (Fig. 1B). Consistent with the reduced oxygen consumption of coa1Δ cells, the mutant cells were compromised for growth on non-fermentable medium containing glycerol/lactate (Fig. 1C). The deletion of COA1 in the BY or W303 backgrounds resulted in an inability to grow on minimal glycerol/lactate medium. However, weak growth could be observed for W303 coa1Δ cells on rich glycerol/lactate medium, whereas BY4743 coa1Δ cells failed to grow (data not shown and Fig. 1C). The addition of copper to rich glycerol/lactate medium enhanced the growth of coa1Δ cells (Fig. 1C). The partial rescue in growth by copper salts was specific for copper, as high concentrations of iron or zinc salts failed to yield cell growth (Fig. 1C).

coa1Δ cells have a specific defect in CcO assembly that is partially rescued by copper

Mitochondria purified from coa1Δ cells exhibit low CcO activity (30% of WT) that can be partially restored by addition of copper to the growth medium (Fig. 2A). Complex II and complex III activities were unaffected by the deletion of COA1 (data not shown). Cells lacking COA1 had reduced steady state levels of CcO subunits Cox1-3 (Fig. 2B) and the level of heme a in mitochondria from coa1Δ cells was only 20% of WT (Fig. 2C) consistent with the low CcO activity. Heme b levels were comparable in mitochondria from coa1Δ or WT cells (Fig. 2C), in accordance with the normal complex III activity.

Since Coa1 was reported to interact with Atp14 in a global two-hybrid interaction study [START_REF] Ito | A comprehensive two-hybrid analysis to explore the yeast protein interactome[END_REF], we assessed the level of complex V in coa1Δ cells. The migration and levels of monomeric and dimeric complex V on Blue Native PAGE (BN-PAGE) were comparable to WT (data not shown), making it unlikely that Coa1 is important for complex V assembly. This selective degradation of Cox1-Cox3 is consistent with an assembly defect in CcO. Taken together, these data show that coa1Δ cells have a post-translational CcO specific defect.

To investigate whether

Coa1 is associated with the IM

Glycerol/lactate growth was fully restored in coa1Δ cells when transformed with 3' Myctagged COA1 on a YCp vector under the control of the MET25 promoter and CYC1 terminator (Fig. 3A). Mitochondria isolated from coa1Δ COA1-Myc cells showed a prominent Coa1-Myc band at the expected mass of 22 kDa (Fig. 3B). The protein is retained in mitoplasts and is not extracted by carbonate buffer (Fig. 3C), low or high salt concentrations and sonication (Fig. 3D) suggesting a tight association with either the inner or outer membrane. Digestion of Coa1-Myc by proteinase K in mitoplasts was slow compared to OM or IMS proteins and was most similar to the degradation pattern of the matrix protein Sod2 (Fig. 3E). Coa1-Myc was degraded if the mitoplasts were initially solubilized with 1% deoxycholate (DOC) or disrupted by sonication, excluding the possibility that Coa1 is somehow resistant to degradation by proteinase K (Fig. 3E).

The hydropathy profile of Coa1 reveals a central, conserved hydrophobic stretch whose length is sufficient to form a transmembrane domain. Overall, these data strongly suggest that Coa1 spans the IM.

MSS51, COX10 and MDJ1 are high copy suppressors of the respiratory defect of coa1Δ cells

To address the role of Coa1 in CcO function, we screened for high copy suppressors of the respiratory growth defect of coa1Δ cells. Cells transformed with a high copy DNA bank prepared from coa1Δ cells were screened for growth on glycerol/lactate medium. From about 200,000 colonies replicated on glycerol/lactate medium, 51 colonies showed significant growth after 5-7 days of incubation at 30°C. The growth of most of these colonies was shown to be vector dependent based on the loss of respiratory growth when the URA3-based vector was shed by 5-fluoroorotic acid selection. Sequencing showed that the plasmids contained different fragments of genomic DNA that encompassed one of three genes. MSS51 was present in 21 plasmids, COX10 in 5 and MDJ1 in 6. MSS51 was a stronger suppressor relative to MDJ1 and COX10 (Fig. 4A).

Due to the low mitochondrial copper in coa1Δ cells (Fig. 1A), we assessed the efficiency of the high copy suppressors upon modulation of the copper concentration in the growth medium.

At 6 mM copper, the suppression of the respiratory defect of coa1Δ by Mdj1 or Cox10 is weakly enhanced (Fig. 4B), but this may arise from the general copper suppression occurring in coa1Δ cells as shown in Fig. 1C. When copper was limited by the chelator bathocuproine sulfonate (BCS), the growth of coa1Δ cells covered with COA1 on a low copy plasmid was slightly attenuated compared to the YPLG medium; however respiratory growth conferred by the high copy MSS51 was more strongly attenuated (Fig. 4B). Thus, the MSS51 suppression of coa1Δ cells appears linked to copper status.

Mdj1 is a DnaJ protein in the mitochondrial matrix that activates the ATPase activity of mtHsp70. Over-expression of mtHsp70 did not rescue the respiratory deficiency of coa1Δ cells, nor did it improve the suppression observed with MDJ1 when the two genes were over-expressed together (data not shown).

Cox10 is the farnesyl transferase in heme a biogenesis, an obligate cofactor bound to Cox1. The suppression by COX10 is dependent on the catalytic function of the Cox10 farnesyl transferase, as a mutant allele that lacked catalytic activity (R212A, R216A) failed to act as a suppressor (data not shown).

MSS51 restores the CcO activity in coa1Δ cells without enhancing Cox1 translation

Mss51 is a multi-function protein acting as a translational activator of COX1 along with Pet309, as a translational elongation factor for COX1 and, in addition, binds the Cox1 nascent chain [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF][START_REF] Perez-Martinez | Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p[END_REF][START_REF] Siep | Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae[END_REF]. MSS51 is a known suppressor of the CcO deficiency in shy1Δ cells by reversing the Cox1 translation defect (Barrientos et al., 2002b). As mentioned, Cox1 translation was normal in coa1Δ cells (Fig. 2D).

Over-expression of Mss51 in coa1Δ cells did not increase COX1 translation in an in vivo mitochondrial translation study during the pulse phase of the reaction (data not shown). Both oxygen consumption measured on intact coa1Δ cells over-expressing Mss51 and CcO activity of mitochondria purified from this strain increased to about 50% of WT (Fig. 5A,B). Steady-state levels of Cox1-3 (Fig. 5C) and heme a levels (Fig. 5D) were also elevated in coa1Δ cells overexpressing Mss51. These data show that over-expression of Mss51 partially reverses the low CcO activity in coa1Δ cells presumably through a post-translational action of binding to nascent Cox1.

Since both Mss51 and Cox10 are related to Cox1 synthesis/maturation, we overexpressed these two proteins together in coa1Δ cells. Although COX10 and MDJ1 are only weak suppressors of coa1Δ cells, COX10, but not MDJ1, had a strong synergistic effect in combination with MSS51 (Fig. 5E). The strong synergism between MSS51 and COX10 suggests that the two jointly correct a defect in coa1Δ cells relating to Cox1 through separate steps.

Shy1 and Coa1 function at a related step of CcO assembly

Given the additive effect of Mss51 and Cox10 co-expression in coa1Δ cells, we tested whether COX10 also had a synergistic effect with MSS51 in shy1Δ cells. The combination of MSS51 and COX10 clearly enhances respiratory growth in shy1Δ (Fig. 6A). However, this phenotype is Mss51-dependent, as over-expression of COX10 by itself could not rescue the respiratory growth defect of shy1Δ cells (data not shown). While COX10 enhanced the stimulatory effect of MSS51 in shy1Δ cells, over-expression of COA1 abrogated it (Fig. 6A).

Over-expression of Coa1 in shy1Δ cells by itself did not diminish the residual CcO activity as assessed by oxygen consumption and CcO activity measurements (data not shown). Therefore, the negative effect of Coa1 seen in shy1Δ cells over-expressing Mss51 is likely due to an impairment of Mss51 function. The level of expression of Coa1 had no effect on the steady state level of Mss51 (data not shown). To determine whether over-expression of Coa1 attenuated the translational stimulation by Mss51 in shy1Δ cells, we evaluated the incorporation of [ 35 S]-Met into mitochondrial translation products. The enhanced translation of Cox1 seen in shy1Δ cells over-expressing Mss51 was unaffected by the over-expression of Coa1 (Fig. 6B), making it likely that Coa1 exerts its negative effect at a post-translational step. Similarly, we assessed whether Shy1 could have a negative effect on the Mss51 suppression of coa1Δ cells. Over-expression of Shy1 neither rescued the respiratory growth defect of coa1Δ cells nor affected the suppression of this defect by high levels of Mss51 (supplemental Fig. 1).

The similarities in residual CcO activity and in MSS51/COX10 suppression of shy1Δ and coa1Δ strains led us to investigate whether shy1Δ cells resembles coa1Δ strains in having a mitochondrial copper defect. shy1Δ cells are deficient in mitochondrial copper (Fig. 6C), yet total cellular copper is normal (data not shown). In addition, supplementation of shy1Δ cultures with exogenous copper partially restores growth on glycerol/lactate medium (Fig. 6D). The copper defect observed in coa1Δ and shy1Δ cells suggested that Cu B site formation could be impaired.

Since Cox11 is implicated in Cu B site formation, we tested whether over-expression of COX11 had a growth stimulatory effect. High copy COX11 neither stimulated respiratory growth of coa1Δ or shy1Δ cells nor was synergistic with MSS51 in suppressing shy1Δ cells (data not shown). Given the similarities between shy1Δ and coa1Δ strains, Shy1 and Coa1 probably act at a closely related step in CcO assembly.

Coa1 is part of a high molecular weight complex and interacts with Mss51

The genetic interaction between COA1 and MSS51 in shy1Δ suggested that the two proteins might interact physically. We constructed a strain in which we integrated 13 copies of the Myc epitope at the 3'end of COA1 and 3 copies of the HA epitope at the 3'end of MSS51.

These tags did not affect the ability of the strain to grow on respiratory medium. Mitochondria isolated from the COA1-13Myc MSS51-3HA cells were solubilized with 0.1% DOC and fractionated on a size exclusion column (Fig. 7A). Analysis of the distribution of Coa1 by immunoblotting of the elution fractions revealed the presence of Coa1-Myc in fractions corresponding to high molecular mass components near 400 kDa. Similarly, BN-PAGE of COA1-13Myc mitochondria revealed the presence of a Coa1 complex of approximately 400 kDa (Fig. 7B).

A fraction of Mss51 was found in a similar mass range as Coa1 (Fig. 7A, complex A in Fig. 7B), in addition to a smaller mass component (B in Fig. 7B). Immunoprecipitation of Mss51-HA from solubilized mitochondria resulted in the co-adsorption of Coa1-Myc demonstrating that the two proteins physically interact (Fig. 7C). Together, these data show that Coa1 and Mss51 are part of a ~400 kDa complex.

Coa1 interacts with Cox14 and nascent Cox1

Mss51 was reported previously to interact with Cox14 [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. To assess whether Coa1 interacts with Cox14 in addition to Mss51, we constructed a strain with 13Myc integrated at the 3' end of COX14 in addition to 3HA integrated on COA1. Immunoprecipitation of Cox14-Myc resulted in the co-precipitation of Coa1-HA (Fig. 8A). Likewise, a strain with a 3HA tag at the 3' end of COX14 in addition to the integrated 13 Myc tag on COA1 enabled the co-precipitation of Cox14 and Coa1 (Fig. 8B). Thus, Coa1 interacts with both Mss51 and Cox14.

Deletion of COX14 disrupted the interaction between Coa1 and Mss51 (Fig. 8C) whereas the interaction between Mss51 and Cox14 was independent of the presence of Coa1 (Fig. 8D). These results suggest that Cox14 may mediate the interaction between Mss51 and Coa1. BN-PAGE confirmed that the 400 kDa Coa1:Mss51 complex is abrogated in cox14Δ cells (Fig. 7B).

Attempts were made to visualize Cox14-Myc after BN-PAGE but were unsuccessful. One possibility is that Cox14 occupies a central position in the complex that may mask the Myc tags, therefore precluding detection by immunoblotting under native conditions.

Since both Cox14 and Mss51 were shown to interact with nascent Cox1 [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF], we tested whether Coa1 also interacts with nascent Cox1. Mitochondrial translation products were labeled in vitro in purified mitochondria from COA1-13Myc or WT cells. The mitochondria were then solubilized in digitonin-containing buffer and the soluble fraction was immunoprecipitated with anti c-Myc-agarose beads. Cox1 was specifically detected in the eluate of the beads incubated with the extracts from COA1-13Myc mitochondria (Fig. 9). This result shows that like Cox14 and Mss51, Coa1 interacts with newly synthesized Cox1.

Coa1 interacts with Shy1 and is necessary for the inhibition of Cox1 translation in shy1Δ cells

The similar phenotypes of coa1Δ and shy1Δ cells suggested that Coa1 might also interact with Shy1. The deletion of COA1 did not affect the protein level of Shy1 and vice versa (data not shown). We constructed a strain with 13Myc integrated at the 3' end of SHY1 in addition to the 3HA tag on COA1. Fractionation of extracts of solubilized mitochondria from this strain showed that Shy1 eluted in fractions similar to those containing Coa1 (supplemental Fig. 2). This result is consistent with the presence of Shy1 in a large molecular weight complex [START_REF] Nijtmans | Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast[END_REF].

Immunoprecipitation of Shy1-Myc resulted in the co-precipitation of Coa1 (Fig. 10A), and the reverse experiment, immunoprecipitation of Coa1-HA led to the recovery of Shy1 with the beads (Fig. 10B) showing that Coa1 and Shy1 are part of a complex. The ~400 kDa Coa1:Mss51 complex observed on BN-PAGE remained intact in shy1Δ cells (Fig. 7B), suggesting that Shy1 is not part of this complex. Shy1 was postulated to disassemble the Mss51:Cox14 complex during normal Cox1 translation and IM insertion [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. As predicted from this model, the abundance of the Coa1:Mss51 complex appears to be enhanced in shy1Δ cells (Fig. 7B).

The attenuated Cox1 translation observed in shy1Δ cells was postulated to arise from stabilization of a Mss51:Cox14 complex that effectively titrates out the Mss51 translational activator [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. Because of the normal translation of Cox1 observed in coa1Δ cells, COA1 was deleted in shy1Δ cells and Cox1 translation was quantified (Fig. 10C). The absence of Coa1 in shy1Δ cells partially reversed the Cox1 translation block. Quantification of the gels from 3 independent in vivo translation experiments showed that only the translation of Cox1 was decreased in shy1Δ cells and that deletion of COA1 in this background significantly restored it, although not to WT levels (Fig. 10D). This result suggests that Coa1 has a role in maintaining Mss51 in a state unavailable for translational activation of Cox1 in a shy1Δ background. Despite the significant recovery of Cox1 translation, the deletion of COA1 in shy1Δ did not reverse the respiratory growth defect or the low mitochondrial copper level (supplemental Fig. 3).

DISCUSSION

One of the many challenges that cells face in building active CcO is to coordinate the assembly of the hydrophobic mitochondrial encoded subunits with the subunits derived from the nuclear genome. This task is even greater when one considers that Cox1 and Cox2 have to acquire their metallic cofactors essential for activity. We have identified the gene COA1 as functioning in linking Cox1 translation and the insertion of its cofactors.

CcO biogenesis in the mitochondrial IM commences with the translation of the mitochondrial encoded subunits mediated by a series of IM-associated translational activators.

These activators appear to recruit mRNA to the IM for co-translational insertion of the nascent polypeptides. Co-translational insertion of Cox1 is dependent on the Pet309 and Mss51 translational activators [START_REF] Manthey | The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae[END_REF][START_REF] Perez-Martinez | Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p[END_REF][START_REF] Siep | Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae[END_REF]. Of the three subunits of CcO encoded by the mitochondrial genome, Cox1 is the first to join the assembly chain. Oxa1 is likely to play a role in the IM insertion of the twelve transmembrane domains of Cox1, as cells lacking Oxa1 show marked Cox1 instability, as seen in a mitochondrial translation assay [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF][START_REF] Bonnefoy | OXA1, a Saccharomyces cerevisiae nuclear gene whose sequence is conserved from prokaryotes to eukaryotes controls cytochrome oxidase biogenesis[END_REF].

Two heme a molecules and a copper atom are buried inside the barrel formed by Cox1 [START_REF] Tsukihara | Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8A[END_REF]. Cox1 has an open channel from the IMS side of the IM where heme a insertion may occur. However, this channel is obstructed by the association of Cox2. It is therefore probable that Cox1 acquires its cofactors at an early stage of the assembly process.

Heme a is coordinated by axial ligands on helices 2 and 10 of Cox1, thus heme a insertion could have a significant effect on the IM folding of Cox1 which could trigger the assembly process to proceed to the next step. An intermediate consisting of Cox1, Cox4 and Cox5A accumulates in human cells from patients carrying mutations in a number of genes encoding assembly factors like Sco1 and SURF1 (human Shy1) [START_REF] Stiburek | Tissue-specific cytochrome c oxidase assembly defects due to mutations in SCO2 and SURF1[END_REF][START_REF] Williams | Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1 or SURF1[END_REF]. The intermediate fails to accumulate in Cox10 or Cox15-deficient patient cells consistent with the idea that heme a insertion occurs prior to the addition of Cox4 and Cox5A (Antonicka et al., 2003a;Antonicka et al., 2003b;[START_REF] Williams | Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1 or SURF1[END_REF].

A clear connection between co-translational insertion of Cox1 and cofactor assembly has not been established to date. We show the gene COA1 is required for efficient CcO assembly in yeast. The respiratory defect of coa1Δ cells is suppressed by high copy MSS51, MDJ1 and COX10 and the suppression by elevated levels of Mss51 and Cox10 is enhanced when the two proteins are co-expressed. This observed synergistic suppression is consistent with the different functions exerted on Cox1 by Mss51 and Cox10 in translation and heme a biosynthesis, respectively. These results suggest that Coa1 links co-translational insertion and heme addition to Cox1 (Fig. 11). Coa1, like Mss51, only has robust homologs within fungi (Supplemental Fig. 4).

However, the presence of a functional homolog that would link translation of Cox1 to the assembly of its cofactors in higher eukaryotes is likely and genetic screens are ongoing in our laboratory to check this possibility. coa1Δ cells resemble shy1Δ cells in many aspects. First, both strains display residual CcO activity that can be suppressed by elevated levels of Mss51. Second, their respiration is enhanced when Mss51 and Cox10 are combined together. Lastly, mitochondria of each strain have low levels of copper. These data and the physical interaction between Coa1 and Shy1 strongly suggest that the two proteins function at a related step of CcO assembly. The enhancement of the respiratory growth of shy1Δ cells over-expressing Mss51 by COX10 is the first indication that Shy1 is linked to a heme a step in eukaryotes. Accordingly, Shy1 appears to be important for insertion/stabilization of the Cu B -heme a 3 in R. sphaeroides [START_REF] Smith | Assembly of cytochrome c oxidase in the absence of the assembly protein Surf1p leads to loss of the active site heme[END_REF].

Mss51 functions in Cox1 translation and elongation of the nascent chain and is known to interact with nascent Cox1 [START_REF] Siep | Mss51p, a putative translational activator of cytochrome c oxidase subunit-1 (COX1) mRNA, is required for synthesis of Cox1p in Saccharomyces cerevisiae[END_REF][START_REF] Perez-Martinez | Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p[END_REF][START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF].

Moreover, Mss51 also interacts with Cox14 [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. Cox1 translation is attenuated in shy1Δ cells and this defect can be rescued either by over-expression of Mss51 or deletion of COX14 (Barrientos et al., 2002b;[START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. Shy1 was postulated to disassemble the Cox1:Cox14:Mss51 complex allowing for Mss51 recycling to its translation/elongation function (Fig. 11) [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. The Cox1:Cox14:Mss51 complex is thought to accumulate in shy1Δ cells, therefore titrating out Mss51 from Cox1 translation/elongation and creating a negative feedback loop that may coordinate translation and membrane insertion [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. The negative feedback loop model may however be more complex as the deletion of SHY1 in cells containing a COX1-ARG8 chimera gave normal levels of the Arg8 reporter [START_REF] Perez-Martinez | Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p[END_REF].

The interaction of Coa1 with Mss51, Cox14 and nascent Cox1 suggests that Coa1 is part of the feedback regulatory loop. Coa1 appears to be a key component of the stalled complex as Cox1 translation defect in shy1Δ cells is partially reversed by deleting COA1 and Cox1 translation is normal in coa1Δ cells. The stalled complex that inhibits Cox1 translation could correspond to the 400 kDa Mss51:Coa1 complex that we detected by BN-PAGE, since we showed that this complex is stabilized in shy1Δ cells and is disrupted upon deletion of COX14 (Fig. 7B). It is likely that the Mss51:Coa1 complex also contains Cox14 and nascent Cox1. We detected a lower molecular weight complex containing Mss51 (Fig. 7A,B). This complex is present at higher level in strains where translation of Cox1 occurs optimally (WT and cox14Δ) than in shy1Δ cells where translation of Cox1 is compromised. Therefore, we suggest that the low molecular weight Mss51 complex may be involved in Cox1 translation.

We propose that Coa1 stabilizes the Mss51:Cox14:Cox1 complex until Shy1 interacts with Coa1, causing Mss51 and Cox14 to disassemble and Cox1 to proceed to the next assembly step involving the insertion of the cofactors (Fig. 11). Even though we showed that the interaction between Mss51 and Cox14 was not dependent on Coa1, the absence of Coa1 will likely preclude the formation of the 400 kDa complex, resulting in normal Cox1 translation. Cells lacking COA1 translate Cox1 normally but are compromised for assembly presumably because Cox1 cannot be presented efficiently to Shy1. The partial suppression of the respiratory defect of coa1Δ cells by over-expression of MSS51 is likely due to the post-translational function of Mss51 which could increase the stability of nascent Cox1 upon binding, allowing eventually more assembly over time. As mentioned, overexpression of MSS51 allows respiratory growth of shy1Δ cells, but the effect is nullified by co-expression of COA1. This dominant negative effect of COA1 on the MSS51-mediated suppression of shy1Δ cells may arise from titrating out a factor such as Cox14 if formation of the Mss51:Cox14:Coa1 complex occurs in a step-wise manner. High levels of COA1 do not titrate out Mss51, as translation of Cox1 is not attenuated in shy1Δ cells over-expressing COA1 and MSS51 (Fig. 6B).

Mdj1 was also recovered as a suppressor of coa1Δ cells. This DnaJ protein associated with mitochondrial Hsp70, may enhance the chaperoning activity of Hsp70. Cells lacking the Mba1 ribosomal receptor or harboring a mutant Oxa1 variant showed an association of Cox1 and Hsp70 [START_REF] Ott | Mba1, a membrane-associated ribosome receptor in mitochondria[END_REF]. Thus, the suppression by Mdj1 in coa1Δ cells may occur through an enhanced chaperoning activity of Hsp70 that may stabilize/refold nascent Cox1 polypeptides stalled for insertion. This effect would be similar to the suppression of coa1Δ by MSS51 discussed above. However, since Mdj1 has been shown to interact with mitochondrial nascent translation products [START_REF] Westermann | Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins[END_REF], a more direct role of Mdj1 in CcO assembly can not be excluded.

Cells lacking Coa1 or Shy1 have mitochondrial copper and respiratory growth defects

that can be partially restored with supplemental copper. Other CcO assembly mutants do not show a similar mitochondrial copper deficiency. The source of copper used for metallation of CcO arises from the matrix copper-ligand complex conserved in eukaryotic cells [START_REF] Cobine | Yeast contain a nonproteinaceous pool of copper in the mitochondrial matrix[END_REF]Cobine et al., 2006a). The observed diminution in matrix copper levels in coa1Δ and shy1Δ cells may suggest that the assembly step at which these two proteins are involved could influence the function of the transporter responsible for trafficking the CuL complex. Studies are underway to identify the copper transporter of the IM.

MATERIALS AND METHODS

Yeast strains and vectors. Yeast strains used in this study are described in supplementary Table I. All FMP deletions screened were in the BY4743 background and were grown in liquid YPD at 30 o C. The coa1Δ strains were in the BY4743 background or in W303 created by disruption using KanMX. Cells were cultured in either rich medium or synthetic complete (SC) medium lacking the appropriate nutrients for plasmid selection. The carbon source used was either 2% glucose or a 2% glycerol-2% lactate. A 3'-Myc tagged version of COA1 was cloned into a yeast low-copy vector under the control of the MET25 promoter and the CYC1 terminator [START_REF] Mumberg | Regulatable promoters of Saccharomyces cerevisiae: Comparison of transcriptional activity and their use of heterologous expression[END_REF]. COA1-Myc was created using PCR, where the 3' oligonucleotide removed the stop codon and added the coding sequence for a 3' Myc tag (LEQKLISEEDL). Sequencing was used to confirm cloning products in all created vectors. Yeast strains were transformed using lithium acetate.

Mitochondria purification and assays. Intact mitochondria were isolated from yeast as described previously [START_REF] Diekert | Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae[END_REF]. Total mitochondrial protein concentration was determined by standard Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Cytochrome c oxidase activity was assessed by monitoring the oxidation of reduced cytochrome c and reaction rates were normalized to total mitochondrial protein [START_REF] Capaldi | Mammalian cytochrome c oxidase: characterization of enzyme and immunological detection of subunits in tissue extracts and whole cells[END_REF]. The oxygen consumption of the cells grown to stationary phase was determined on a 5300A Biological Oxygen Monitor (Yellow Springs Instrument Co.). Only the linear response was considered in calculating the rate of oxygen consumption [START_REF] Horng | Human Sco1 and Sco2 function as copper-binding proteins[END_REF]. Metals were quantified in isolated mitochondria using a Perkin-Elmer Optima 3100XL inductively coupled plasma optical emission spectrometer (ICP-OES) after nitric acid digestion at 95 o C. A standard curve was determined from commercially available standards (Perkin-Elmer), and buffer samples were run in parallel as a control. Heme analysis was conducted on 1-2 mg of mitochondria extracted with 0.5 mL of acetone containing 2.5% HCl as described previously [START_REF] Barros | Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O[END_REF]. The pH of the extract was adjusted to 4 by addition of 1 μL formic acid and titration of a KOH solution. The sample was clarified by centrifugation at 13000 rpm for 5 min and 1 mL was injected onto a 3.9 x 300 mm C18 Bondclone column (Phenomex, USA).

In vitro and in vivo mitochondrial protein synthesis. Purified mitochondria were labeled in vitro as described [START_REF] Westermann | Analysis of mitochondrial translation products in vivo and in organello in yeast[END_REF]. For in vivo labeling, the cells were grown overnight in selective medium containing 2% raffinose and then re-inocculated in YP-2% raffinose to grow to an absorbance of 1 at 600 nm. The labeling and preparation of the samples for 12% SDS-PAGE was as described in (Barrientos et al., 2002b). The gel was dried and radiolabeled proteins were visualized by exposing autoradiographic films at -80°C.

Blue native gel electrophoresis (BN-PAGE).

BN-PAGE was performed essentially as described [START_REF] Wittig | Blue native PAGE[END_REF] except 1.5% digitonin was used. After incubation for 20 min on ice and centrifugation (20000g for 10 min at 2 o C) supernatants were mixed with sample buffer (5% Coomassie brilliant blue G250, 0.5 M 6-aminocaproic acid, pH 7.0) and loaded on 4-13% gradient polyacrylamide gel. Separated complexes were detected by immunoblotting on PVDF membrane.

Immunoblotting. Proteins samples were separated on 15% acrylamide gels and transferred to nitrocellulose. ECL reagents were used to visualize proteins with horseradish peroxidaseconjugated secondary antibodies. Anti-Myc and HA antisera were purchased from Santa Cruz, anti-porin was from Molecular Probes and antisera to Cox1-Cox3 were from Mitosciences.

Antiserum to Sod2 was provided by Dr. Val Culotta, and antisera to Cyb2, Cyt1 and Cyc1 provided by Dr. Carla Koehler.

Localization Experiments. Proteinase K treatment was done as previously described [START_REF] Diekert | Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae[END_REF]. Carbonate extraction experiments used mitoplasts made by diluting 125 μg intact mitochondria 20-fold in 10 mM Hepes, pH 7.4. Mitoplasts were then incubated in 100 mM sodium carbonate, pH 11.3 for 30 min on ice and centrifuged at 100000g. The supernatant and pellet were analyzed by immunoblotting. High salt extraction experiments also used mitoplasts that were sonicated three times for 30 sec in 25 mM, 100 mM or 500 mM NaCl.

Construction of the high copy library. 36 μg of genomic DNA purified from coa1∆ cells was partially digested with Sau3A. DNA fragments between 3 and 10 kb were purified from a 1% agarose gel. The plasmid YEP352 was digested to completion with SalI. The plasmid DNA was then treated with Klenow polymerase in the presence of dCTP and dTTP while the genomic DNA was treated with dATP and dGTP. The resulting plasmid DNA was treated with 1 unit of calf intestinal alkaline phosphatase for 30 min at 37°C. Ligation of YEP352 with the genomic DNA was conducted overnight at 16°C. The total number of colonies obtained was estimated to be about 20,000, representing a theoretical 5-fold coverage of the yeast genome.

Gel Filtration. Mitochondria (0.3-0.5 mg) were resuspended in solubilization buffer (PBS, 0.1% DOC). After incubation on ice for 10 min, lysates were centrifuged at 16000 g for 25 min at 4 o C, passed through a 0.45 μm filter and loaded onto a Superdex 200 (GE Healthcare) column equilibrated in solubilization buffer. Note: Supplementary information is available at the EMBO J. Online. Protoporphyrin peaks (P) were found to vary as reported previously [START_REF] Barros | Regulation of the heme a biosynthetic pathway in Saccharomyces cerevisiae[END_REF]. D) In vivo labeling of mitochondrial translation products. coa1∆ and BY4743 cells were pulsed for 15 min with 35 S-methionine at which point 20 mM of cold methionine was added. The reaction was either stopped (P) or allowed to chase (C) for 90 min at 30°C. The samples were run on 12% SDS-PAGE, the gel was dried and exposed to autoradiographic film. The same cells as in A) were spotted on rich media (YP) supplemented with 50 μM FeCl 2 containing 2% glycerol-2% lactate (YPLG) or 2% glucose (YPD) and either the copper specific chelator BCS or CuSO 4 . The plates were incubated at 30°C for 2 days (YPD) or 6 days (YPLG).

REFERENCES FIGURE LEGENDS

Fig. 5. Restoration of CcO by high copy MSS51 in coa1∆ cells. A) coa1∆ cells with high copy

MSS51 or an empty vector (vec) were grown in SC-2% raffinose and assayed for oxygen consumption. B) Mitochondria were purified from these cells and assayed for CcO activity. C) Immunoblot of 30 μg of mitochondria from coa1∆ cells containing an empty vector (vec), a low copy vector expressing COA1 or a high copy vector expressing MSS51. D) Heme profile from 2 mg of mitochondria purified from coa1∆ containing high copy MSS51 or an empty vector. A/B represents the ratio of the area under the curve for the peaks of heme b (B) and heme a (A). E) coa1∆ cells expressing high copy MSS51 and containing an empty vector (vec) or high copy vectors with MDJ1 or COX10 were grown in SC-2% raffinose, serially diluted and spotted on SC 2% glycerol-2% lactate or 2% glucose with selection. The plates were incubated at 30°C for 2 days (glucose) or 7 days (glycerol-lactate). Mitochondria isolated from either wild-type (WT), shy1Δ or cox14Δ strains containing genomically tagged COA1 (COA1-13Myc, lanes 1-3) or MSS51 (MSS51-13Myc, lanes 4-6) were solubilized in buffer containing 1.5% digitonin. Lysates were loaded onto a continuous 4-13% gradient gel and protein complexes were separated by BN-PAGE. The distribution of complexes was analyzed by immunoblotting with mouse monoclonal anti-Myc antibody. Monomeric (V 1 ) and dimeric (V 2 ) forms of respiratory chain complex V served as a control and were visualized using antisera against F 1 -subunit. C) 0.3 mg of mitochondria from COA1-13Myc or COA1-13Myc MSS51-3HA cells were solubilized in Tris 20 mM pH 7.4, 100 mM NaCl, 1 mM PMSF, 0.1% lauryl maltoside and clarified extracts were immunoprecipitated with rabbit polyclonal HA antiserum and protein A agarose beads. The load representing 5% of the extracts and the entire fraction of the last wash and bead eluate were analyzed by immunoblotting. [START_REF] Barrientos | Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae[END_REF]. This ternary complex is recruited by Coa1 and likely forms the ~400 kDa complex that we detected by BN-PAGE. Membrane insertion of Cox1 probably occurs via Oxa1. Shy1 joins the complex and promotes the dissociation of Cox14 and Mss51 that is recycled to its translational function. The cofactor insertion in Cox1 can then proceed via Cox10, Cox15 (heme a biosynthesis) and Cox11 (Cu B insertion) leading to holo-Cox1.

  Coa1 has a role in the translation of mitochondrial encoded subunits of CcO, [ 35 S]-Met incorporation into mitochondrial translation products was monitored in vivo. coa1Δ cells exhibit a labeling profile of mitochondrial encoded proteins similar to WT cells after the 15 min pulse (Fig 2D). During the 90 min chase, the levels of Cox1-Cox3 diminished markedly in coa1Δ cells, whereas Var1 and Cyt b remained stable as in the WT cells.

Immunoprecipitation. 0

 0 .3-0.5 mg of purified mitochondria were solubilized by incubation in 0.5 mL Tris 20mM pH 7.4, 100 mM NaCl, 1mM PMSF and either 0.1% lauryl-maltoside or 1% digitonin (IP buffer) for 30 min at 4 o C. Lysates were centrifuged at 16000 g for 15 min at 4 o C. Immunoprecipitation was performed by incubating overnight at 4 o C the clarified lysates with 40 μL anti-Myc or HA rabbit polyclonal antisera. The antibodies were precipitated by the addition of 20 μL of recombinant protein A agarose beads (Pierce) for 4 h at 4 o C. The beads were pelleted by centrifugation at 2000 g for 2 min and washed with 0.8 mL of IP buffer for 5 min. This step was repeated 3-4 times. Beads were eluted by boiling for 5 min in 50 μL SDS-PAGE loading buffer. The clarified extracts and wash fractions were TCA precipitated prior to electrophoresis and immunoblotting. Statistical procedures. Descriptive statistics are represented as average ± standard error. The unpaired student t-test (two tail) was used to compare differences between experimental and control groups. Acknowledgements. This work was supported by a grant ES 03817 from the National Institutes of Environmental Health Sciences, NIH to D.R.W. We acknowledge the support of the CEMH core facility for FPLC chromatography (DK P30 072437) and United Mitochondrial Disease Foundation for support for P.A.C. We acknowledge Dr. Peter Rehling for open discussions on Coa1.

Fig. 1 .

 1 Fig. 1. coa1Δ cells are respiratory deficient and have low mitochondrial copper. A) Copper

Fig. 2 .

 2 Fig. 2. Deletion of COA1 results in a CcO specific defect. A) coa1∆ cells and the isogenic WT

Fig. 3 .

 3 Fig. 3. Mitochondrial localization of Coa1. A) coa1Δ cells expressing a low copy version of

Fig. 4 .

 4 Fig. 4. High copy suppressors of the respiratory defect of coa1∆ cells. A) coa1∆ cells

Fig. 6 .

 6 Fig. 6. High copy suppression of shy1∆ cells and low mitochondrial copper levels. A) shy1∆

Fig. 7 .

 7 Fig. 7. Coa1 and Mss51 form similar sized complexes and interact. A) Extracts of 0.5 mg of

Fig. 8 .

 8 Fig. 8. Coa1 interacts with Cox14 which is required for the Coa1:Mss51 interaction. A) 0.3

Fig. 9 .

 9 Fig. 9. Coa1 interacts with newly translated Cox1. Purified mitochondria from WT cells or cells containing a chromosomally tagged COA1-13Myc were labeled with 35 S-Met for 20 min.

Fig. 10 .

 10 Fig. 10. Coa1 interacts with Shy1 and deletion of COA1 relieves the repression of Cox1 translation in shy1Δ cells. A) 0.3 mg of mitochondria from COA1-3HA or COA1-3HA SHY1-

Fig. 11 .

 11 Fig. 11. Model for Coa1 linking Cox1 translation to cofactors assembly. After the translation