Ottilie Von Loeffelholz

Verena Kriechbaumer

Richard A Ewan

Rafal Jonczyk

Susann Lehmann

Jason C Young

Ben M Abell
email: b.abell@shu.ac.uk

OEP61 is a chaperone receptor at the plastid outer envelope

Keywords: OEP61, chaperone receptor, Hsp70, tail-anchored protein, protein targeting, plastid

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Introduction

Organelles require proteins that are encoded by nuclear DNA, necessitating protein delivery from the cytosol. Generally, this targeting process is promoted by cytosolic factors, which increase specificity and efficiency, and the best understood processes are initiated by N-terminal targeting signals on the precursor protein [START_REF] Wickner | Protein translocation across biological membranes[END_REF]. Targeting signals can also be provided by internal transmembrane domains (TMDs), which are more hydrophobic than N-terminal targeting sequences. Precursor proteins with either type of targeting signal can be bound by molecular chaperones to promote their delivery to organellar membranes.

Although the Hsp70 and Hsp90 chaperones assist protein folding, they can also work with other cytosolic factors to promote organellar targeting. A general mechanism of the chaperones is to protect hydrophobic parts of precursors so that they are maintained in an unfolded, targeting compatible state. Hsp70 is widely involved in targeting processes, and it often functions as part of a complex. For example, the targeting of chloroplast proteins bearing an Nterminal targeting sequence is promoted by Hsp70 in combination with a 14-3-3 protein, which is known as the guidance complex [START_REF] May | 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants[END_REF]. Other proteins bound for the chloroplast are assisted by Hsp90 [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF], or by an ankyrin-repeat protein AKR2A [START_REF] Bae | AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis[END_REF][START_REF] Dhanoa | Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope[END_REF]. Similar targeting mechanisms exist for mitochondrial protein targeting, which in the case of inner membrane proteins is promoted by Hsp70 and Hsp90 [START_REF] Fan | Hsp90 functions in the targeting and outer membrane translocation steps of Tom70mediated mitochondrial import[END_REF][START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF][START_REF] Zara | Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90[END_REF] and the Hsp70-regulatory co-chaperone Hsp40 [START_REF] Bhangoo | Multiple 40-kDa heat-shock protein chaperones function in Tom70-dependent mitochondrial import[END_REF]. Disruption of Hsp70 or Hsp90 activity by specific inhibitors or mutation of a chaperone binding site reduces targeting of the precursors to mitochondria. Also, the mitochondrial targeting of some matrix proteins are assisted by the peptidylprolyl isomerase AIP/XAP2 [START_REF] Yano | AIP is a mitochondrial import mediator that binds to both import receptor Tom20 and preproteins[END_REF]. Although many proteins destined for the ER membrane are targeted cotranslationally by SRP (reviewed by [11]), posttranslational targeting of tail-anchored membrane proteins can be mediated by Hsp70 and Hsp40 [12,[START_REF] Rabu | A precursorspecific role for Hsp40/Hsc70 during tail-anchored protein integration at the endoplasmic reticulum[END_REF] as well as the ASNA1/TRC40/Get3 targeting factor (reviewed by [START_REF] Abell | Tail-anchored membrane proteins: exploring the complex diversity of tail-anchored-protein targeting in plant cells[END_REF]). The posttranslational targeting of some yeast ER proteins is also promoted by Hsp70 and Hsp40 [15].

Chaperones are able to deliver precursors to organelles via membrane bound chaperone receptors. These receptors interact with chaperones via their 'TPR clamp' domain, comprising three TPR motifs to form a peptide-binding groove of seven -helices [START_REF] Brinker | Ligand discrimination by TPR domains. Relevance and selectivity of EEVDrecognition in Hsp70 x Hop x Hsp90 complexes[END_REF]. Specific interaction occurs with the highly conserved Cterminal ends of Hsp70 or Hsp90, which contain carboxyl groups capable of forming ionic bonds with the amino groups within the TPR domain [START_REF] Brinker | Ligand discrimination by TPR domains. Relevance and selectivity of EEVDrecognition in Hsp70 x Hop x Hsp90 complexes[END_REF][START_REF] Scheufler | Structure of TPR domainpeptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine[END_REF]. Binding of Hsp70 requires at least a heptapeptide (PTIEEVD), while the last five amino acids (MEEVD) of Hsp90 are essential and sufficient for binding [START_REF] Brinker | Ligand discrimination by TPR domains. Relevance and selectivity of EEVDrecognition in Hsp70 x Hop x Hsp90 complexes[END_REF][START_REF] Scheufler | Structure of TPR domainpeptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine[END_REF][START_REF] Wu | Different combinations of the heat-shock cognate protein 70 (hsc70) C-terminal functional groups are utilized to interact with distinct tetratricopeptide repeat-containing proteins[END_REF] At least one TPR containing receptor can be found at each organelle [START_REF] Schlegel | The tetratricopeptide repeats of receptors involved in protein translocation across membranes[END_REF], suggesting a universal role for chaperone receptors in protein targeting. The most prominent examples for chaperone receptors are Tom70 at human and yeast mitochondria [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF], and Toc64 at plant chloroplasts [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF]. Both receptors are proposed to mediate early recognition of chaperone bound precursor proteins A c c e p t e d M a n u s c r i p t 4 via their TPR domain. Targeting information is recognized by a separate domain on Tom70 before transfer of the precursor complex to the translocase [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF], or in the case of Toc64, by other receptors that are in closer proximity to the translocase [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF]. When Tom70 is knocked out in combination with another mitochondrial receptor, Tom20, yeast cells are not viable, even though neither single knockout shows a lethal phenotypic defect [20]. This indicates a functional overlap between Tom70 and Tom20, and may explain difficulties in confirming a phenotypic effect of Toc64 knockout in A. thaliana [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF]21,22]. In support of this notion, Toc64 was found to interact only with precursors bound to Hsp90 and not to bind the guidance complex containing Hsp70 [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF]. Therefore, other chaperone receptors may exist at the chloroplast to accept precursors bound to Hsp70.

To identify other potential chaperone receptors we performed a database search for proteins containing a clamp-type TPR domain and a TMD. This resulted in the identification of an uncharacterised protein in A. thaliana termed OEP61, which has a TPR clamp domain at its N-terminus and a single TMD at its C-terminus. We show that OEP61 is expressed throughout the mature plant, and localises to the outer envelope of chloroplasts. OEP61 specifically binds Hsp70, and can selectively recognise precursors destined for the chloroplast. Furthermore, the soluble portion of OEP61 is able to inhibit the chloroplast targeting of precursors. Therefore, we propose that OEP61 is a novel chaperone receptor involved in the targeting of chloroplast precursors from the cytosol.

DNA constructs

The coding sequence of A. thaliana OEP61 (clone pda11784 from RIKEN) was inserted into pSPUTK (Stratagene). Sequences encoding truncated versions of A. thaliana OEP61 were cloned into pET-16b (Novagen) after amplification by PCR using the following primers: 5'-ATATCTCGAGTTTAACGGGTTAATGGATCC-3' and 5'-ATATAGATCTTTATTTTCCGAACAACCACTTC-3' for OEP61-TM (amino acids 1-534), 5'-ATATAGATCTTTATTTTCCGAACAACCACTTC-3' and 5'-ATTAAACATATGGAGACAATTGCCGATGTG-3' for L (amino acids 214-534), 5'-ATTAAACTATGTATCAGATCAATGCAGCTC-3' and 5'-TTAAATTGGATCCTATGCCTTGCCAGGTCC-3' for TPR (amino acids 103-213). The R185A mutation was introduced into OEP61-TM by PCR corresponding to the mutation used in Tom70 (R192A for human Tom70 and R171A for yeast Tom70; [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF]) using the following primers: 5'-GGAATGTCAAAGCCCTATACGCAAGGGGTCAAGCTTACAGA-3' and 5'-TCTGTAAGCTTGACCCCTTGCGTATAGGGCTTTGACATTCC-3'. The clone for TPR1 of human Hop (HopTPR1) is described in [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF]. Plasmid DNA for standard curves in qRT-PCR of OEP61 was inserted into pSPUTK with the following primers (5'-ATATAGATCTACCATGTTTAACGGG TTAATG-3' and 5'-ATATAGATCT CTAGTT TCCAATATAGCC-3'), and plasmid DNA for the endogenous control was donated from Erich Glawischnig [START_REF] Schuhegger | Regulatory variability of camalexin biosynthesis[END_REF]. Plasmids for the pulldown of precursor proteins were obtained from TAIR, which was also used to assign subcellular localisation. A plasmid for Lhcb1 (clone AB80 from pea; [START_REF] Cashmore | Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide[END_REF]) was kindly donated by Colin Robinson. For the removal of targeting sequences, PCR templates were generated to delete residues 268-297 of Toc33, and to delete residues 1-54 of pSSU.

Plant material

A. thaliana Col-0 (Nottingham Arabidopsis Stock Centre) plants were grown on soil with 14 h light of 100 mol m -2 s -1 at 21°C with 60% humidity. Adult plant tissue was harvested after 8 weeks.

Transient expression system

Four-week-old tobacco plants (Nicotiana tabacum SR1 cv Petit Havana) were inoculated via their lower epidermal surface with Agrobacterium strain GV3101 (pMP90) bearing an expression vector, using the method described by [START_REF] Batoko | A rab1 GTPase is required for transport between the endoplasmic reticulum and golgi apparatus and for normal golgi movement in plants[END_REF].

Confocal imaging

A Zeiss LSM 510 laser scanning microscope was used with a 63 oil immersion objective (Zeiss), as described in [START_REF] Kriechbaumer | Subcellular distribution of tail-anchored proteins in Arabidopsis[END_REF]. WCIF ImageJ software was used to test for colocalisation. Pixels of the red and green channels of 8-bit images are classed as colocalised if their intensities are higher than the threshold of their channels (default settings 50) and if the ratio of their intensity is higher than the ratio setting value (default settings 50%).

Quantitative RT-PCR

Total RNA was extracted from 50-100 mg plant tissue using Trizol® reagent (Invitrogen) according to the manufacturers' instructions. After treatment with DNAse (NEB), cDNA was synthesized using random primers (Invitrogen) and M-MuLV reverse Transcriptase (NEB) according to the manufacturers' instructions. Quantitative RT-PCR was performed with the standard curve experiment on the StepOne Real-Time PCR System (Applied Biosystems) using Sybr Green PCR master mix and the following intron spanning primer sets for OEP61 and actin: 5'-CTGGAAAGTTCTGATTGCTTC-3' and 5'-CATCAAGAGGTGTGGTGATTG-3' for OEP61 and 5'-TGGAACTGGAATGGTTAAGGCTGG-3' and 5'-TCTCCAGAGTCGAGCACAATACCG-3' for actin. PCR products were analysed on agarose gels after amplification, and their sequences determined to confirm the products. Quantifications of OEP61 mRNA in different tissues were calculated in relation to actin.

Immunoblotting

Total protein was extracted from plant tissue by grinding frozen plant material in extraction buffer (50 mM Tris pH8.0, 100 mM KOAc, 1 mM EDTA, 1mM DTT, 20% glycerol, 1% Triton X-100, 1% SDS, 1% plant protease inhibitor (Sigma)). Insoluble parts were separated from the extract by centrifugation. Protein concentrations were measured with detergent compatible Bradford Ultra (Expedeon) reagent using bovine serum albumin as a standard, and equal amounts of total protein were used for immunoprecipitation. Prior to immunoblotting, OEP61 was immunoprecipitated from total protein extracts. Samples were precleared by adding four volumes of TXIP buffer (10 mM Tris-HCl pH7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton, 2 mM PMSF) and 0.1 volume pansorbin (Calbiochem) and incubated at 4°C for 30 min. The mixture was centrifuged at 14300 g for 10 min at 4°C to pellet the pansorbin, and the supernatant was incubated with anti-OEP61-IgG at 1:1000, followed by over night incubation at 4°C. Protein A sepharose beads (Sigma, 1:100) were added and incubated for 1.5 h at 4°C. The beads were washed 4 times with 1 ml TXIP buffer, denaturing loading buffer was added, and eluted proteins were separated by SDS-PAGE. For immunoblotting, proteins were transferred from SDS polyacrylamide gels to PVDF membranes (Millipore) according to the manufacturers' instructions. Primary antibodies were used at 1:10000 for the anti human Hsp70 IgG (Stratagene) and 1:1000 for the anti-OEP61-IgG serum (Eurogentec, Brussels, Belgium). Anti-OEP61 has been optimised for buffer pH and dilution and evaluated against recombinant Toc64, OEP61, and BSA protein and pre-immune serum as well as small, large and final bleed were tested in pulldown experiments with radiolabeled OEP61 (Figure S2). The secondary goat anti-rabbit IgG labeled with red-fluorescent Alexa Fluor 594 dye (Invitrogen) or green-fluorescent IRDye 800CW, respectively was used at 1:3000 and signals were detected using the ODYSSEY Infrared imaging system (LI-COR Biosciences).

For immunoblotting of OEP61 in A. thaliana, Col-0 plants were used to prepare chloroplasts as described for pea plants used in competitive targeting assays [START_REF] Kriechbaumer | Subcellular distribution of tail-anchored proteins in Arabidopsis[END_REF]. Thermolysin treatment was performed with half of the chloroplasts using 40 U/ml thermolysin for 5 min at 30°C. 30 μl of chloroplast fractions with and without thermolysin treatment and the supernatant fraction from the initial chloroplast fractionation were immunoblotted using anti-OEP61-IgG.

Transcription and translation

Proteins of interest were fused to the pSPUTK SP6-promoter via overlapping extension-PCR [START_REF] Urban | A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR[END_REF]. Transcriptions were performed with 15 μg PCR-fusion product and SP6-RNA-Polymerase (New England Biolabs) according to the manufacturer's manual. Protein translations were performed in wheat germ extract (Promega) according to the manufacturer's instructions using Easy Tag Express 35S (Perkin Elmer).

Binding experiments

Proteins were expressed in T7 Express I q E.coli cells (NEB), and purified by chromatography on Ni-NTA agarose (Promega). Pulldown experiments with recombinant protein or from WGE, respectively, were performed according to [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF] with the following changes: 1 M His-tagged protein was bound in buffer CG (100 mM KOAc, 20 mM HEPES-KOH [pH 7.5], 5 mM MgOAc 2 ) containing 2 mg/ml ovalbumin and 0.1% Triton X-100. Incubation of the matrix bound recombinant proteins occurred in buffer CG containing 2 mg/ml ovalbumin, 0.1% Triton X-100 and 25% WGE. The decapeptide GAGPKIEEVD mimicking the C-terminus of plant Hsp70 was used as a competitive inhibitor in a final concentration of 500 M. Buffer CG containing only 0.1% Triton X-100 was used for washing steps. Radiolabeled precursor proteins were synthesised by in vitro translation in WGE (Promega), treated with 1 unit/ml apyrase for 5 min on ice (NEB) and subjected to the same binding assay.

Competitive targeting assay

The competitive targeting assay for OEP61 was performed according to [START_REF] Kriechbaumer | Subcellular distribution of tail-anchored proteins in Arabidopsis[END_REF]. Briefly, chloroplasts from pea leaves and mitochondria from maize coleoptiles were incubated with prespun in vitro translated protein and incubated at 30°C for 20 min. Organelles were repurified by two successive centrifugation steps and a wash with 0.1 M Na 2 CO 3 . Both fractions, and an import sample treated with 40 U/ml thermolysin for 5 min at 30°C, were analysed by SDS-PAGE and Cyclon Phosphor Screen (Packard). ER import was performed cotranslationally for 15 min with 50 A 280 U ml -1 dog microsomes. Microsomes were pelleted in a TLA100.2 fixed angle rotor (Beckman) and 180000 g for 45 min and proteins visualised by SDS-PAGE and Cyclon Phosphor Screen (Packard). For inhibition

Phylogenetic Analysis

The protein sequence of the TPR domain from OEP61 was compared to proteins in public databases in three ways: a) using BLAST against a non redundant protein database; b) using BLAST against the non redundant protein database with the organisms restricted to A. thaliana; and c) compared to TPR domains from A. thaliana homologues of known chaperone receptors and Hop (Sti1). Homologues of A. thaliana chaperone binding TPR domains were manually selected using PROSITE (http://www.expasy.org/prosite/). Similar sequences to the TPR domain of OEP61 were identified using BLAST. Duplicates were omitted and protein sequences were aligned using the ClustalW program (Align-m) using default settings changing the output format setting to PHYLIP (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Unrooted trees were generated with PHYLIP on the web (http://bioweb2.pasteur.fr/phylogeny/intro-en.html) using the programmes protdist, seqboot (included in protdist, using 100 replicates), neighbour, consense (included in neighbour, using 100 replicates) and drawtree. Bootstrap values and names were inserted manually.

Accession numbers

Locus identifiers and ABRC clone numbers are given in brackets: OEP61 (At5g21990); Toc33 (At1g02280, 190I17); Toc34 (At5g05000, 167B21); Tic22 (At4g33350, 144A17); At2g17 (At2g17972.1, U50887); ANT homologue (ANTh, At5g56450, 285A11); Tom22-I (At1g04070, 136E14); Tim9 (At3g46560, 135L1); Tim10 (At2g29530, U23564); At5g013 (At5g01340, M73L24, mitochondrial substrate carrier); pSSU (At1g67090, U13397, ribulose bisphosphate carboxylase small chain 1A); and Lhcb1 (clone AB80 from pea).

Results

Search for chaperone receptors

To identify potential chaperone receptors a sequence search was designed using the consensus from characterised TPR clamp proteins. Structural alignment was performed for the TPR clamp region of proteins Hop, FK506binding protein 5, FK506-binding protein 4, cyclophilin-40, serine/threonine phosphatase 5, cyclophilin seven suppressor, Tom70, Tom34, and Unc-45. This alignment produced degenerate motifs for the two clamp motifs that are required to support chaperone binding, and these motifs were used to scan the protein databases Swiss-Prot, TrEMBL and TrEMBL New [23]; this resulted in the identification of nine novel proteins. For such a protein to act as a receptor, it would also require anchorage to a membrane, so the potential for at least one TMD was used to refine the search. A single uncharacterised protein in A. thaliana was found to contain both a TPR clamp domain and a predicted transmembrane domain, which we have named OEP61. Figure 1 shows a scheme of the OEP61 structure and the alignment of its TPR domain with other TPR clamp proteins used to generate the consensus sequence. OEP61 homologues can be identified in plants with available sequence data, including rice and algae, but homologues could not be identified in other types of organisms. There are high sequence similarities between these homologues in the TPR domain and the C-terminus of the proteins, although the "linker region" between the TPR clamp and the TMD is much more variable (Figure S1). This indicates that the function and localisation elements are conserved and that OEP61 has the potential to act as a chaperone receptor in plant cells. In line with our classification of OEP61, an independent bioinformatics approach to detect proteins with TPR clamp domains also detects OEP61 (termed AtTPR7) as part of a group of 24 uncharacterised predicted proteins in A. thaliana [START_REF] Prasad | In silico identification of carboxylate clamp type tetratricopeptide repeat proteins in Arabidopsis and rice as putative co-chaperones of Hsp90/Hsp70[END_REF].

Phylogenetic analysis of OEP61

To gain insight into the potential function of OEP61, relationships between the TPR domain of OEP61 and other TPR clamp proteins were determined by phylogenetic comparisons of protein sequences. Initially, TPR domains from different organisms were investigated by using BLAST to find similar sequences. The resulting tree contains four functionally distinct families: the TPR domains of PPIases; stromal membrane-associated protein 1 (SMAP-1 / Unc-45); Tom34; and unknown protein products (Figure S2A). Tom70 and Hop are more distantly related, and hence are not included. The PPIase group is the largest group in the tree and includes cyclophilins and FK506 binding proteins (FKBPs), of which FKBP52, FKBP38 and cyclophillin40 are known to bind Hsp90 [30]. Hsp90 binding has also been shown for the C-terminal TPR domain of Tom34 [START_REF] Young | Specific binding of tetratricopeptide repeat proteins to the C-terminal 12-kDa domain of hsp90[END_REF], which is a chaperone receptor at the mitochondrial outer membrane in deuterostomia [START_REF] Schlegel | The tetratricopeptide repeats of receptors involved in protein translocation across membranes[END_REF][START_REF] Chewawiwat | Characterization of the novel mitochondrial protein import component, Tom34, in mammalian cells[END_REF]. OEP61 is in a branch together with unknown plant proteins, which show high sequence similarities to the full sequence of OEP61, indicating that OEP61 is part of a novel, uncharacterised protein family in plants. When the analysis was restricted to relationships between OEP61 and TPR clamp proteins in A. thaliana a similar pattern emerges (Figure S2B). Four families are represented in the tree: Toc64; Hsp70 interacting proteins; TPR domain containing DnaJs; and PPIases. OEP61 is most similar to the TPR domains of PPIases, but is not located inside this group, suggesting that OEP61 is not directly related to any characterised TPR clamp proteins.

To focus on the relationships of the TPR domain, sequences from A. thaliana homologues of known chaperone receptors were collected manually and used for phylogenetic analysis (Figure S2C). The resulting phylogenetic tree divides the TPR domains into four distinct groups: The three different TPR domains of Hop build a branch each and the fourth branch is built by the TPR domains of three different Toc64 homologues, Toc64-III, Toc64-V and an A. thaliana homologue of the human fatty acid amide hydrolase (F13N6) [START_REF] Pollmann | Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana[END_REF] and MGLN6, a fourth uncharacterised protein, which contains a TPR domain with high similarity to the TPR domain of Toc64. OEP61 is part of the Toc64 branch, showing that it is more similar to Toc64 than to any of the Hop TPR domains, although the TPR domains of Toc64 [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF] and its mitochondrial counterpart, mtOM64 [START_REF] Lister | Functional definition of outer membrane proteins involved in preprotein import into mitochondria[END_REF], are more closely related to each other than to the TPR domain of OEP61.

OEP61 is expressed in all tissues

To test the distribution of OEP61 in mature A. thaliana plants, expression levels of OEP61 in different tissues were measured using qRT-PCR and immunoblotting (Figure 2). OEP61 mRNA could be detected in all tissues, indicating that OEP61 is ubiquitously expressed, although levels of OEP61 varied from 25% to 190% compared to actin (Figure 2A). The highest transcription levels were detected in leaf tissues, and the lowest in roots. These results differs slightly from the expression pattern derived for Toc64, which has similar mRNA levels in buds, rosette leaves and roots [21].

Since regulation at the posttranscriptional level may occur (e.g. [START_REF] Carthew | Origins and Mechanisms of miRNAs and siRNAs[END_REF], immunoblots using an antibody raised and tested (Figure S3) against recombinant OEP61 were performed (Figure 2B). To reduce interference from abundant proteins such as rubisco, OEP61 protein was immunoprecipitated prior to immunoblotting. OEP61 was detected in all tested tissues, suggesting a general role for OEP61. Higher OEP61 protein levels were found in green tissue, including leaves and buds, which correlates with the transcript analysis. The highest protein level was detected in young leaves, suggesting that OEP61 plays an important role during leaf growth.

OEP61 is localised at the chloroplast outer envelope

The restriction of OEP61 homologues to plants suggested that OEP61 has a plant specific function and may be localised to plastids. According to sequence analysis OEP61 lacks an N-terminal, cleavable signal peptide (ChloroP: no chloroplast transit peptide detected, score: 0.457; SignalP-HMM signal peptide probability: 0.00) and has a single TMD of 22 amino acids at the C-terminus by TMHMM [START_REF] Krogh | Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[END_REF]. These features define OEP61 as a tail-anchored (TA) protein, a class of membrane proteins that depend on their TMD and flanking sequences for targeting and membrane insertion [START_REF] Rabu | Biogenesis of tail-anchored proteins: the beginning for the end?[END_REF]. TA proteins are localised to most internal membranes of cells, including ER, mitochondria, and chloroplasts. It is possible to predict localisation in plant cells from the sequence of the tail anchor, although this is not entirely reliable, and is particularly difficult for plastidial proteins [START_REF] Kriechbaumer | Subcellular distribution of tail-anchored proteins in Arabidopsis[END_REF]. The sequence analysis resulted in a predicted localisation in the chloroplast, so this was tested by transient expression of YFP-OEP61 in tobacco leaves (Figure 3A). Here, the fluorescence of YFP-OEP61 co-localises with the chlorophyll autofluorescence (indicated with arrows) and GFP-tagged Toc33 (Figure S4), showing localisation of OEP61 to chloroplasts whereas no overlap in fluorescence could be observed with proteins localised to other organelles, including ER and mitochondria (Figure S4). To investigate targeting without the potential interference of tags, OEP61 was synthesised by in vitro translation and incubated with purified organelles, as previously performed to analyse the subcellular distribution of TA proteins [START_REF] Kriechbaumer | Subcellular distribution of tail-anchored proteins in Arabidopsis[END_REF]. In the presence of both chloroplasts and mitochondria, the majority of OEP61 associates with the chloroplast fraction (Figure 3B). No change in molecular weight is detected, confirming the predicted lack of a cleavable targeting sequence. Washes of the organelles included sodium carbonate, proving strong evidence that OEP61 is inserted into the membrane of the outer envelope. Furthermore, OEP61 was unable to insert into ER membranes (data not shown). Therefore, the in vitro assays provide further support for an exclusive localisation of OEP61 to chloroplasts.

Since OEP61 is predicted to be a TA protein, the N-terminus of the protein was expected to be exposed to the cytosol, thereby maintaining a membrane topology similar to the chloroplast TA proteins Toc33 and Toc34 [START_REF] Qbadou | Membrane insertion of the chloroplast outer envelope protein, Toc34: constrains for insertion and topology[END_REF]. Thus, topology was tested by addition of the protease thermolysin after incubation of OEP61 with chloroplasts, and comparisons were made with Tic22 (intermembrane space protein) and Lhcb1 (thylakoid protein) (Figure 3C). Protease addition resulted in digestion of OEP61 (Figure 3C, lane 2), indicating that most of the protein is outside the chloroplast. Tic22 and Lhcb1 remain intact (Figure 3C, lane 4 and 6), confirming that the chloroplasts were impermeable to protease. The tail-anchor of OEP61 is too small to be detected efficiently, although we can infer that the transmembrane domain is inserted into the outer envelope due to its resistance to sodium carbonate washes, which were included in the fractionation of chloroplasts from mitochondria. A comparable response to protease treatment is observed for the plastidial TA protein Toc33 (Figure S5). The topology of OEP61 in its native environment was confirmed by its protease sensitivity in A. thaliana chloroplasts (Figure 3D). We conclude that OEP61 is a TA protein localised exclusively to chloroplasts. Its topology results in the TPR domain facing the cytosol, consistent with a role as a chaperone receptor.

The TPR domain of OEP61 binds specifically to Hsp70

The presence of a TPR clamp domain indicated that OEP61 would bind to Hsp70 and/or Hsp90. To investigate this potential interaction, purified recombinant OEP61 lacking its TMD (OEP61-TM; Figure 4A) was incubated in wheat germ extract and bound proteins were co-precipitated. Compared with a mock incubation (Figure 4B, lane 1), an additional protein band appeared at approximately 70 kDa (Figure 4b) and was shown to be Hsp70 via Western Blotting (Figure S6), suggesting that OEP61 binds to Hsp70. To test whether this interaction was mediated by the TPR clamp domain, we tested the impact of the mutation R185A in OEP61-TM, which is equivalent to the R192A mutation in human Tom70 known to disrupt binding to molecular chaperones [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF]. This mutation abolished binding to the 70 kDa band, showing that OEP61 binds to Hsp70 using its TPR clamp domain. Another band of approximately 25 kDa was pulled down by both forms of OEP61-TM, which indicates an interaction that is not dependent on the TPR clamp. The identity of this protein is not known.

The binding of OEP61 to Hsp70 suggests that there is a specific recognition of Hsp70 and an absence of Hsp90 binding. This was tested by incubating Histagged OEP61 with purified recombinant A. thaliana Hsp70 (At5g02500) [START_REF] Lin | Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana[END_REF] and the major constitutive Hsp90, termed Hsp81 (At5g56030) [40]. Figure 4c shows that His-OEP61 is able to bind Hsp70 efficiently, but does not bind Hsp81. This contrasts with the ability of the His-tagged TPR2A domain of Hop (His-Hop2A) to bind Hsp81 and not Hsp70. Since Hsp70 and Hsp81 were purified using His tags, we verified that their tags had been efficiently removed and did not contribute to their own pulldown by incubating Hsp70 and Hsp81 with beads alone (lanes 7-10). We conclude that OEP61 is able to discriminate between Hsp70 and Hsp81.

To investigate the nature of the interaction between OEP61 and Hsp70 we performed in vitro binding assays in wheat germ lysate with various domains (Figure 4A): OEP61TPR is the TPR domain alone; and OEP61L is the 34 kDa linker sequence between the TPR domain and the tail anchor. The N-terminal TPR domain of human Hop (HopTPR1) is known to bind the C-terminal end of Hsp70 specifically [START_REF] Scheufler | Structure of TPR domainpeptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine[END_REF], and was therefore used as positive control for Hsp70 binding. A strong Hsp70 signal was detected by immunoblot when a TPR domain was present (Fig. 4D, lanes 2, 3 and 5). OEP61-TM (lane 3) has a stronger affinity to Hsp70 than HopTPR1 (lane 2), which could be due to sequence dissimilarities between human and plant Hsp70. Comparison between the binding capacities of OEP61TPR and OEP61-TM show that the TPR domain binds Hsp70 less efficiently than the complete cytosolic domain of OEP61 (47% in lane 5 compared to 100% in lane 3). This suggests that the long linker region between the TPR domain of OEP61 and the transmembrane domain is necessary to maintain the structural stability of a functional TPR domain. Hsp70 binding by OEP61L (lane 4) and OEP61R185A (lane 6) does not exceed background levels of binding obtained by beads alone (lane 1), indicating that the binding of OEP61-TM to Hsp70 is mediated by the wellcharacterised interaction between the TPR clamp and the C-terminus of Hsp70, with no direct contribution from the linker region.

Addition of a peptide representing the C-terminus of the plant Hsp70 strongly inhibited binding to Hsp70 (lanes 8 and 9), with a similar effect on both OEP61 and HopTPR1. This further supports the conclusion that the interaction between OEP61 and Hsp70 is mediated via the TPR clamp domain. Hsp70 binding by the TPR domain of OEP61 alone is not inhibited by the peptide, suggesting that this interaction occurs due to the chaperone activity of Hsp70 acting on misfolded OEP61; this provides further evidence that the linker domain provides stability to the TPR domain.

OEP61 associates selectively with chloroplast precursors

Localisation of OEP61 at the outer chloroplast envelope and interaction of its cytosolic TPR domain with Hsp70 indicates that OEP61 is a plastidial chaperone receptor. To investigate whether it plays a role in protein targeting analogous to Toc64, the interaction of OEP61 with cytosolic precursor complexes was investigated. Precursor proteins were synthesised by in vitro translation in wheat germ extract, and chaperone binding to the precursor proteins was stabilised by incubation with apyrase. These complexes were tested for their ability to bind OEP61-TM and its inactive form OEP61R185A (Figure 5A). The chloroplast precursors of Toc33, Toc34 and Lhcb1 bind specifically to the native form of OEP61. In contrast, the mitochondrial precursors Tom22-1, ANT-h, and Tim9, and Tim10 do not bind to OEP61. Pulldown by both the native and mutated forms of OEP61 is observed for the chloroplast protein Tic22, and the mitochondrial protein At5g013, indicating an interaction that is independent of the TPR clamp. One chloroplast precursor, At2g17, does not interact with OEP61. Taken together, the emerging pattern is that OEP61 is able to interact specifically with some chloroplast precursors, but not with mitochondrial precursors. This pattern holds for different types of precursors, including those in the outer membranes and those translocated to different internal compartments. The requirement for an intact TPR clamp domain suggests that the interaction with precursors is mediated via Hsp70 in the lysate. To determine whether OEP61 binds the targeting sequence of precursors the C-terminal tail anchor of Toc33 was deleted, and the N-terminal targeting sequence of pSSU was deleted. Both of these deletions abolished binding to OEP61 (Figure 5B). Therefore, these data support our general proposal that OEP61 is involved in protein targeting to chloroplasts.

If OEP61 coordinates the transfer of precursors to the translocase of the chloroplast outer envelope, we reasoned that a soluble form of OEP61 lacking its membrane anchor (OEP61-TM) would inhibit targeting. Therefore, the effect of OEP61-TM on the targeting of Toc33 to chloroplasts was tested by in vitro assay (Figure 6A). Toc33 targeting was severely impaired by OEP61-TM (lanes 1 and 2), but was unaffected by the addition of the peroxisomal targeting factor PEX19 (lanes 3 and 4). No mistargeting to ER occurred (lanes 5 and 6). The targeting of a mitochondrial protein At3g58840 to mitochondria was unaffected by OEP61-TM (translation lane 7; lanes 10 and 11), and no mistargeting to chloroplasts was observed (lanes 8 and 9). Similarly, targeting of the ER protein Sec61 to ER microsomes was unaffected by OEP61-TM (lanes 12 and 13). These data show that OEP-TM selectively inhibits chloroplast targeting, without affecting precursors destined for mitochondria or the ER. This is consistent with The inhibition of chloroplast targeting by OEP61 could have arisen primarily due to its ability to bind Hsp70, so we have also tested the ability of HopTPR1 to inhibit chloroplast targeting. Figure 6B shows that the targeting of Toc33, Lhcb1, and pSSU to chloroplasts is severely impaired by OEP61, but is not significantly affected by HopTPR1. Therefore, we conclude that some of the capacity for OEP61 to recognise precursors occurs independently of Hsp70.

Discussion

The need to deliver cytosolic proteins to different organelles has resulted in numerous pathways that use various targeting factors and their cognate membrane-bound receptors. Since chaperones are found to bind many precursor proteins, the importance of chaperone receptors is gaining prominence. Chaperone receptors have been identified at the outer membranes of every organelle [START_REF] Schlegel | The tetratricopeptide repeats of receptors involved in protein translocation across membranes[END_REF], and have been shown to increase the efficiency of protein targeting [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF][START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF]. To understand their role in the fidelity of targeting, and gain a systematic understanding, a more complete knowledge of chaperone receptors is necessary. Here we identify OEP61 as a novel chaperone receptor at the chloroplast outer envelope.

We show that OEP61 is part of an uncharacterised protein family expressed ubiquitously in the plant. It inserts into the chloroplast outer envelope via its tail anchor, exposing the N-terminal TPR clamp domain to the cytosol. In vitro experiments show that the TPR clamp domain of OEP61 interacts specifically with Hsp70, and together with the linker domain facilitates selective recognition of chloroplast precursor complexes. Non-anchored OEP61 interferes with chloroplast targeting. Thus, we propose that OEP61 is involved in protein targeting to chloroplasts, in a manner analogous to the Hsp90-mediated recognition of precursors by Toc64.

OEP61 is expressed throughout the plant, which supports a constitutive role for OEP61 in protein targeting to the chloroplast. The expression pattern is similar for transcripts and protein, with greater levels in green tissues, which is consistent with the localisation of OEP61 to chloroplasts. However, detection of OEP61 in root tissues shows that OEP61 is also likely to reside in root plastids. There are no major differences in distribution compared to Toc64, which is also found throughout the plant [21]. The elevated levels of OEP61 in young leaves indicate a role in chloroplast biogenesis since this is dependent on high levels of protein import. Overall, it is clear that OEP61 exists in all tissues at substantial levels, and is likely to function in all plastids.

A crucial feature of OEP61 to support its potential function in protein targeting is the C-terminal TMD, which ensures a cytosolic disposition of the TPR clamp domain. This domain structure is classed as tail-anchored, and these proteins invariably possess a topology with the N-terminus in the cytosol [START_REF] Rabu | Biogenesis of tail-anchored proteins: the beginning for the end?[END_REF]. TA proteins are involved in many protein targeting processes, such as: Toc33 and Toc34 in the chloroplast translocase [41]; Tom5, Tom6 and Tom7 in the mitochondrial translocase [42]; and Sec61 , Sec61 and RAMP4 in the ER translocase [11,[START_REF] Pool | A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase[END_REF]. Therefore, the TA protein classification of OEP61 is consistent with a role in delivering proteins to the chloroplast translocase.

The TPR domain of OEP61 specifically binds Hsp70 and interacts preferentially with chloroplast precursors. The simplest scheme for the interaction between OEP61 and chloroplast precursors is sequential binding mediated by Hsp70, in which hydrophobic motifs within the precursor bind to Hsp70, and then the C- A c c e p t e d M a n u s c r i p t terminus of Hsp70 binds OEP61 (Figure 8). Chloroplast precursors are known to bind Hsp70 [START_REF] May | 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants[END_REF], yet the widespread interactions of Hsp70 with proteins localised throughout the cell implies that a more sophisticated recognition process takes place. This could occur via additional contacts between OEP61 and the precursor, possibly involving the linker region of OEP61. Such an interaction is mediated by additional TPR motifs in Tom70 [44], but in OEP61 no identifiable functional domains other than the TPR clamp can be identified by sequence homology. Nevertheless, the ability of purified OEP61 to distinguish between chloroplast and mitochondrial precursors, and the inability of HopTPR1 to substitute for OEP61 in the inhibition of chloroplast targeting, suggests that OEP61 is able to directly recognise targeting signals. Alternatively, OEP61 might act in concert with additional receptors such as Toc64. A precedent for the coordination of multiple receptors is the action of multiple Tom70s in mitochondrial targeting [START_REF] Wiedemann | The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria[END_REF], and the identification of large chloroplast precursor complexes suggests that multiple chaperones may be bound to the same precursor [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF]. Such a scheme potentially provides great flexibility to the recognition process, and could combine roles for Hsp70 and Hsp90 in the targeting of a single precursor. More complexity is possible if other precursor receptors exist in the outer envelope. One source of such receptors could be other A. thaliana TPR clamp proteins, which have been identified by an independent bioinformatics analysis, providing a further 23 uncharacterised genes, some of which generate multiple transcript species by alternative splicing [START_REF] Prasad | In silico identification of carboxylate clamp type tetratricopeptide repeat proteins in Arabidopsis and rice as putative co-chaperones of Hsp90/Hsp70[END_REF].

Another possible mechanism for generating targeting specificity is the specific recognition of individual Hsp70 family members, a concept that is supported by specialisation of function of the Hsp70 SSA family in yeast [START_REF] Sharma | Function of SSA subfamily of Hsp70 within and across species varies widely in complementing Saccharomyces cerevisiae cell growth and prion propagation[END_REF]. A. thaliana possesses at least 18 distinct Hsp70 proteins [START_REF] Lin | Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana[END_REF]. Com70 is an Hsp70 in spinach that binds to chloroplasts and associates with translocating precursors [47]; if a chloroplast-bound Hsp70 exists in A. thaliana OEP61 may coordinate its activity. Although such schemes allow a role for chaperone receptors in the specificity of targeting, it is still possible that they simply provide a mechanism for removing chaperones from the precursor to prepare it for recognition by other receptors and for translocation across the membrane. This issue will need to be addressed by manipulating chaperone receptors in plants or in assays with multiple organelles.

Another issue raised by this study is the functional relationship between OEP61 and Toc64. Toc64 has been shown to interact dynamically with the Toc complex in pea [START_REF] Sohrt | Toc64, a new component of the protein translocon of chloroplasts[END_REF], thereby positioning it at the core of targeting and translocation processes, and it has the capability of interacting with precursors bound to Hsp90 [START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF], yet its depletion in T-DNA knockout A. thaliana and in Physcomitrella does not lead to any major phenotypic effect [21,22]. Potentially, OEP61 binds the same precursors as Toc64, since mitochondrial precursors are known to form multi-chaperone complexes that include both Hsp70 and Hsp90 [START_REF] Young | Molecular chaperones Hsp90 and Hsp70 deliver preproteins to the mitochondrial import receptor Tom70[END_REF], and different chloroplast precursors are known to be bound by Hsp70 or Hsp90 [START_REF] May | 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants[END_REF][START_REF] Qbadou | The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64[END_REF]. Such overlap in function may reduce the dependency on Toc64, and thereby explain the difficulty of assessing the A c c e p t e d M a n u s c r i p t functional role of Toc64. Functional redundancy has been observed for the mitochondrial receptors Tom20 and Tom70, in which deletion has a synthetic phenotypic effect [20]. Manipulation of OEP61 and Toc64 in parallel would provide an opportunity to assess the role of both receptors. The spatial relationship between OEP61 and the Toc complex will be an important feature of any coordinated action, and remains to be elucidated. The ability of the nonanchored OEP61 to interfere with targeting suggests that the coordination with other membrane components is vital for the function of OEP61. Overall, the widespread tissue expression and subcellular colocalisation suggests that OEP61 coexists with Toc64 in the outer envelope of plastids, and together they potentially facilitate the targeting of precursors bound to both Hsp70 and Hsp90.

Several targeting factors are involved in chloroplast localisation, and it will be important to understand how they operate alongside the chaperone-mediated system. The guidance complex contains Hsp70, so may interact with OEP61 [START_REF] May | 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants[END_REF]41]. AKR2A has recently been characterised as a cytosolic interaction partner of chloroplast membrane proteins [START_REF] Bae | AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis[END_REF], and has been shown to be involved in the targeting of Toc33 and Toc34 [START_REF] Dhanoa | Distinct pathways mediate the sorting of tail-anchored proteins to the plastid outer envelope[END_REF]. AKR2A is therefore expected to compete with Hsp70 for binding to the hydrophobic TMD, and several independent targeting pathways may operate. Determining the preferred targeting pathways for different classes of chloroplast precursors will require a large-scale systematic analysis, and can be expected to show significant redundancy between pathways.

Here, we have shown that OEP61 is a novel plastidial chaperone receptor for Hsp70, with potential to promote the selective targeting of plastidial precursors. Further studies will be required to determine the overall role for OEP61 in protein localisation, and its relationship with the Toc complex. B. Competitive targeting assay. OEP61 was synthesised by in vitro translation in wheat germ extract (WGE, lane 1, 10% input) and incubated with purified chloroplasts and mitochondria. The chloroplasts (lane 2) and mitochondria (lane 3) were repurified and washed with sodium carbonate before analysis by SDS-PAGE. C. Topology of OEP61 at chloroplasts. Radiolabeled, in vitro translated protein was incubated with purified pea chloroplasts, which were pelleted and washed with sodium carbonate, followed by thermolysin proteolysis in lanes 2, 4, and 6. Tic22 resides in the intermembrane space and Lhcb1 resides in the thylakoids. Molecular weight markers are shown in kDa. D. Topology of native OEP61 at chloroplasts. A. thaliana chloroplasts were isolated and washed with sodium carbonate prior to treatment of equal quantities with (+Th) or without thermolysin, and then immunoblotted alongside supernatant and recombinant OEP61-TM. OEP61 may be involved in the early recognition of Hsp70-bound precursors, in parallel with the recognition of Hsp90-bound precursors by Toc64, and it is also possible that precursors may engage both Toc64 and OEP61. Binding of the chaperone by the chaperone receptor would deliver the targeting sequence of the precursor to Toc34 (or Toc33) and promote release of the molecular chaperones. The precursor would then become translocated across the outer

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  Journal Immediate Publication. Published on 25 May 2011 as manuscript BJ20110448 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110448 A c c e p t e d M a n u s c r i p t 5 Experimental Identification of OEP61 Alignment of known TPR clamp domains was used to generate semi stringent motifs consisting of [K-(ETNDK)-(KQEIR)-(GA)-(NT)-(DEVKT)-(AYFCL)-(YF)] for clamp 1 and [K-(AG)-(YFL)-(YFT)-R-(KR)-(GA)-(AEQK)] for clamp 2, and loose motifs consisting of [(KR)-(ETNDKALQGD)-(LKQEIHSA)-(GA)-(NKT)-(DAELSVNHQKT)-(ACFYLKHQMS)-(YFLV)] for clamp 1 and [K (AGVC)-(YFL)-(AYFTSN)-(RQ)-(IKRQL)-(GAS)-(NATEQKLCS)] for clamp 2. These motifs were used to scan the protein databases Swiss-Prot, TrEMBL and TrEMBL New [23].

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  Journal Immediate Publication. Published on 25 May 2011 as manuscript BJ20110448 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110448 A c c e p t e d M a n u s c r i p t 14 OEP61 functioning to select precursors for delivery to the chloroplast translocase.

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Figure 1 :

 1 Figure 1: OEP61 relationships with other TPR clamp proteins. A. Schematic structure of OEP61 (Swiss-Prot accession number Q8GWM6). The N-terminal TPR domain is defined by PROSITE and a C-terminal transmembrane domain is predicted by SMART between amino acids 531 and 553. The alignment file shows the TPR domain of OEP61 with the known chaperone binding TPR clamp domains from Hop (hHop1 and hHop2A; P31948), FK506-binding protein 5 (hFKBP5; Q13451), FK506-binding protein 4 (hFKBP4; Q02790), cyclophilin-40 (hCyc40; Q08752), serine/threonine phosphatase 5 (hSTP5; P53041), cyclophilin seven suppressor (scCyc7s; A6ZL97), Tom70 (hTom70; O94826), Tom34 (hTom34; Q15785), and Unc-45 (hUNC45A; Q9H3U1).

Figure 2 .

 2 Figure 2. OEP61 is expressed throughout the mature plant.A. RNA levels of OEP61 relative to actin were measured in rosette leaves, cauline leaves, stems, roots, buds, flowers, and siliques from adult plants by quantitative real time PCR. B. Immunoblot against immunoprecipitated OEP61 from young leaves (2 weeks after germination), and rosette leaves, cauline leaves, stems, roots, buds, flowers, and siliques from adult plants. Recombinant OEP61 lacking its TMD (OEP61-TM) is used as a comparison.

Figure 3 .

 3 Figure 3. OEP61 is localised to the outer envelope of chloroplasts. A. Confocal microscope image of YFP-tagged OEP61 construct transiently expressed in tobacco leaves. Its subcellular localisation is overlaid with chlorophyll autofluorescence. Areas of significant colocalisation (by ImageJ) are shown in white.B. Competitive targeting assay. OEP61 was synthesised by in vitro translation in wheat germ extract (WGE, lane 1, 10% input) and incubated with purified chloroplasts and mitochondria. The chloroplasts (lane 2) and mitochondria (lane 3) were repurified and washed with sodium carbonate before analysis by SDS-PAGE. C. Topology of OEP61 at chloroplasts. Radiolabeled, in vitro translated protein was incubated with purified pea chloroplasts, which were pelleted and washed with sodium carbonate, followed by thermolysin proteolysis in lanes 2, 4, and 6. Tic22 resides in the intermembrane space and Lhcb1 resides in the thylakoids. Molecular weight markers are shown in kDa. D. Topology of native OEP61 at chloroplasts. A. thaliana chloroplasts were isolated and washed with sodium carbonate prior to treatment of equal quantities with (+Th) or without thermolysin, and then immunoblotted alongside supernatant and recombinant OEP61-TM.

Figure 4 .

 4 Figure 4. OEP61 interacts specifically with Hsp70 via its TPR clamp domain. A. Variants of OEP61 constructs: OEP61FL (aa 1-554; full length); OEP61TPR (aa 103-213); OEP61L (aa 214-534); OEP61-TM (aa 1-534); OEP61R185A (point mutation of amino acid 185 from arginine to alanine in OEP61-TM).

Figure 5 .

 5 Figure 5. OEP61 associates specifically with precursors destined for the chloroplast via its TPR clamp domain. A. Radiolabeled precursors of Toc33, Toc34, Lhcb1, Tic22, At2g17, ANT homologue (ANT-h), Tom22-I, Tim9, Tim10, and At5g013 were synthesised in wheat germ lysate, treated with apyrase, and then incubated with His-tagged OEP61-TM or the mutated form OEP61R185A. Products bound to His-tagged protein and pulled down with Ni-NTA beads were analysed by SDS-PAGE. Loading was adjusted to correct for the translation efficiency of each protein. Full length products are indicated by a black square, specific pulldowns are indicated by a star, and non-specific pulldowns are indicated with a circle. Sizes are given in kDa. B. Pulldown by OEP61-TM was also performed for Toc33 and pSSU with and without their targeting sequences (TS).

Figure 6 .

 6 Figure 6. Soluble OEP61 inhibits chloroplast targeting. A. Radiolabelled precursors were synthesised by in vitro translation and incubated with chloroplasts or ER membranes (Toc33 and Sec61 ), or chloroplasts and mitochondria in a competitive assay (mitoAt5g58), in the presence of OEP61-TM or PEX19. The organelles were pelleted and analysed by SDS-PAGE. Molecular weight markers are shown in kDa. B. Chloroplasts targeting assays were performed for Toc33, Lhcb1, and pSSU in the presence of OEP61-TM, the Hsp70-binding domain of Hop (HopTPR1), or PEX19.

Figure 7 .

 7 Figure 7. Proposed scheme of OEP61 and Toc64 action.OEP61 may be involved in the early recognition of Hsp70-bound precursors, in parallel with the recognition of Hsp90-bound precursors by Toc64, and it is also possible that precursors may engage both Toc64 and OEP61. Binding of the chaperone by the chaperone receptor would deliver the targeting sequence of the precursor to Toc34 (or Toc33) and promote release of the molecular chaperones. The precursor would then become translocated across the outer

Biochemical

  Journal Immediate Publication. Published on 25 May 2011 as manuscript BJ20110448 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110448 A c c e p t e d M a n u s c r i p t

Figure 1 Biochemical

 1 Figure 1

* 5 A

 5 Figure 3 A

  Figure 6

Figure 7 Biochemical

 7 Figure 7

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgements

We thank ABRC, RIKEN, Chris Hawes, Alison Baker, Erich Glawischnig, Colin Robinson and Peter Cain for supplying plasmids. Thanks to Chris Hawes' group for advice with in vivo localisation, and to Robert Mullen for discussions on the manuscript.

Funding This work was supported by BBSRC Research Grant awarded to BMA [BB/E01559X/1]. JCY holds a Canada Research Chair in Molecular Chaperones, Tier II.

envelope using established mechanisms involving Toc159 and the pore formed by Toc75. OEP61 and Toc64 would then dislocate from the core complex.

Titles of supplementary figures


Wiebke Neumann
email: wiebke.neumann@vfm.slu.se

Göran Ericsson
email: goran.ericsson@vfm.slu.se

Holger Dettki
email: holger.dettki@vfm.slu.se

W Lutz

Does off-trail backcountry skiing disturb moose?

Keywords: Experimental disturbances, Human impact, Individual-based analysis, Alces alces, Sweden

The advancement of recreational activities into wildlife habitat calls for a better knowledge about the effects of human-induced disturbances, particularly in systems where humans dominate wildlife mortality. We exposed nine adult free-ranging female moose repeatedly to off-trail backcountry skiing to study moose behavior and habituation using a controlled field experiment in Northern Sweden. Moose response was short-term, but distinct. Moose moved 33-fold faster during the first hour following disturbance, resulting in almost a doubling of the energetic usage per kilogram body weight. Movement rates increased 3 h following disturbances, came along with enlarged activity ranges at the day of disturbance, and resulted in moose leaving the original area. We found no evidence for habituation. Because of the short-term response, the effect of single skiing disturbance events on the overall energy budget of large-bodied animals in good body condition is likely to be negligible. We recommend off-trail skiers to avoid following wildlife tracks because such disturbances bear risk for more severe consequences on wildlife's energy budget if wildlife resists habituation, if an animal's risk perception is high, or when the frequency of disturbance increases.

Introduction

Human recreational activities expand increasingly into wildlife habitat, becoming a part of wildlife's environment [START_REF] Ingold | Freizeitaktivitäten im Lebensraum der Alpentiere[END_REF][START_REF] Knight | Wildlife and recreationists: coexistence through management and research. Island, Washington Lavsund S, Sandegren F[END_REF][START_REF] Stankowich | Ungulate flight responses to human disturbance. A review and meta-analysis[END_REF]. Human-induced disturbances may affect wildlife ecology because they may trigger a behavioral response analog to predation risk, generating similar behavioral costs [START_REF] Picton | Energetic cost of wildlife displacement by winter recreationists[END_REF][START_REF] Frid | Human-caused disturbance stimuli as a form of predation risk[END_REF][START_REF] Fortin | Behavioural response of free-ranging bison to human disturbance[END_REF][START_REF] Reimers | Behavior responses of wild reindeer to direct provocation by snowmobile or skier[END_REF][START_REF] Taylor | Wildlife response to recreational and associated visitor perceptions[END_REF]. In areas where human harvest accounts for the major source of wildlife mortality, the impact of human activity on wildlife is of particular interest (e.g., [START_REF] Ericsson | Age-specific moose Alces alces mortality in a predator-free environment: evidence for senescence in females[END_REF][START_REF] Lima | Life in a multipredator environment-some considerations for anti-predatory vigilance[END_REF][START_REF] Solberg | Age-specific harvest mortality in a Norwegian moose Alces alces population[END_REF]. Antipredator behaviors are complex [START_REF] Liley | What best explains vigilance in elk: characteristics of prey, predators, or the environment?[END_REF]. Predation risk affects animals not only directly, but also indirectly by changing animal's behavior such as their spatial organization, resulting in the "landscape of fear" and causing trophic cascades [START_REF] Berger | Fear, human shields and the redistribution of prey and predators in protected areas[END_REF][START_REF] Brown | Foraging and the ecology of fear[END_REF][START_REF] Creel | Relationships between direct predation and risk effects[END_REF]. Compared to on-trail activity, the unpredictable temporal and spatial character of off-trail activities often result in a larger influence zone [START_REF] Taylor | Wildlife response to recreational and associated visitor perceptions[END_REF][START_REF] Stankowich | Ungulate flight responses to human disturbance. A review and meta-analysis[END_REF]). In Northern Europe, off-trail activities such as backcountry skiing, hiking, and snowmobile driving are popular recreational activities. Besides the type of disturbance, animal's activity prior disturbance, relative location to the origin of disturbance, and topographic features further influence the level of disturbance [START_REF] Cassirer | Elk responses to disturbances by cross-country skiers in Yellowstone National Park[END_REF][START_REF] Reimers | Behavior responses of wild reindeer to direct provocation by snowmobile or skier[END_REF][START_REF] Stankowich | Ungulate flight responses to human disturbance. A review and meta-analysis[END_REF].

However, animals that do not relocate in the presence of disturbance may not imply undisturbed wildlife, but a lack of alternative habitats to switch to [START_REF] Gill | Why behavioural responses may not reflect the population consequences of human disturbance[END_REF]. Like predation risk, human disturbances can cause increased levels of stress affecting physiological parameters [START_REF] Creel | Snowmobile activity and glucocorticoid stress responses in wolves and elk[END_REF]. Impact on physiological conditions such as increased stress levels or movement activity caused by disturbance may bear severe consequences for wildlife that range in seasonal environments and that must handle conservative energy management strategies [START_REF] Schwartz | Nutrition and energetics[END_REF]. Locomotion costs vary with animal size and ground cover, and smaller animals such as, e.g., moose calves face relatively greater locomotion costs, especially when moving in snow [START_REF] Coady | Influence of snow on behaviour of moose[END_REF][START_REF] Fancy | Incremental cost of activity[END_REF].

Using a controlled field experiment and state-of-the-art Global Positioning System (GPS) technology, we studied the impact of off-trail backcountry skiing activity with following up of moose tracks on moose behavior. The degree of habituation may vary among individuals in a population [START_REF] Picton | Energetic cost of wildlife displacement by winter recreationists[END_REF][START_REF] Stankowich | Ungulate flight responses to human disturbance. A review and meta-analysis[END_REF]. Thus, we applied individual-based behavioral analyses to test for habituation using moose movement rates, i.e., "speed" (in meters per hour), as a proxy and analyzed moose daily activity ranges to address the impact on moose spatial organization.

Based on theory and previous research, we predict that disturbance by off-trail backcountry skiing would be reflected by (1) an increase in moose average movement rates, (2) an increase in moose diurnal activity area, and (3) disturbance would result in a spatial reorganization of moose, i.e., moose activity ranges would overlap less after disturbance than before. Based on humans being the predominant source of moose mortality in our study area, we also predicted that (4) moose would not habituate to the repeated, but unpredictable, human-induced disturbance stimuli. In addition, we estimated the metabolic costs associated with the predicted increased movement rates for adults and potentially following moose calves.

Material and methods

We collected GPS location data from nine adult free-ranging female moose during March 6-11 in 2006. Moose ranged between the inland boreal forests to the low alpine areas in Northern Sweden with an average moose density of around 0.3 individuals per square kilometer, excluding the areas above tree line (Fig. 1). During winter, moose ranged in the inland forest (64°26′ N, 19°22′ E; WGS84) that is dominated by monocultures of Scots pine Pinus sylvestris covering a gently rolled landscape with an average elevation of 309 m±87 standard deviation (SD). Human density is moderate with an average of 6.0 humans per square kilometer and 1.0 km road per square kilometer (Statistics Sweden 2008;Swedish Land Survey 2008). With the start of the vegetation period, moose migrate to the low alpine area (65°42′ N, 16°46′ E; WGS84) that consists of boreal and mountainous forest, partly above tree line, with an average elevation of 996 m±424 SD, low human density (0.9 humans per square kilometer; Statistics Sweden 2008) and relatively low accessibility (0.4 km road per square kilometer; Swedish Land Survey 2008).

Swedish moose populations experience a sustained hunting pressure with hunting as the major source of moose mortality that accounts for 81% of mortality in female moose [START_REF] Ericsson | Age-specific moose Alces alces mortality in a predator-free environment: evidence for senescence in females[END_REF]Lavsund and Sandegren 1989;Swedish AHWM 2009). In contrast, the mortality risk from natural predators is low, particularly for adult moose (Swedish EPA 2008;[START_REF] Swenson | Predation on moose calves by European brown bears[END_REF]).

Moose disturbances

We immobilized moose from a helicopter using a dart gun to inject a mixture of an anesthetic and a tranquilizer (etorphine and xylazine; [START_REF] Arnemo | Risk of capturerelated mortality in large free-ranging mammals: experience from Scandinavia[END_REF]. We equipped each female with a collar that included a GPS receiver, Global System for Mobile (GSM) communication modem, and a traditional very high frequency (VHF) beacon (Vectronic Aerospace GmbH, Berlin, Germany). The GPS collars calculated a position at least every 30 min. The GPS collar sent the information onto a database server using the GSM cell phone network [START_REF] Dettki | Real-time moose tracking: an Internet based mapping application using GPS/GSM-collars in Sweden[END_REF].

Using the last known GPS position followed by traditional VHF tracking, we approached the free-ranging moose directly as allowed by the terrain by one to two persons on cross-country skis that moved silent. The female moose were prime aged and we assumed all individuals to be non-naïve because they ranged at least partly, i.e., during nonparturition time, within human proximity where crosscountry skiing and other recreational activities are common.

After initial disturbance by approaching human on crosscountry skies and displacement of the moose, we performed an additional tracking for 30 min to address the impact of tracking. To study if moose habituate to disturbances, we repeated the treatments three times for each moose individual with 2 days between repetitions. To evaluate the effect of reproductive status, we included barren females (n=3) as well as females accompanied by offspring (n=6).

At the time and position of disturbance, we recorded temperature, precipitation type and strength, wind strength and direction, habitat data, and snow condition as well as the extent to which moose sank in relation to snow depth. To access moose perception of risk and stress level, we recorded if the moose individual fled immediately when encountered, first defecated before flight, or left calmly the position of disturbance.

Data analysis

Movement activity and daily activity range

For each moose, we calculated the distance in meters from the previous position using Euclidean distance and computed its movement rate (in meters per hour). To access individual relative moose response, we extracted the speed in the hours following disturbance and compared it with the individual's speed during the respective hour on the previous day, i.e., 24 h before, using Wilcoxon signed ranks test. This allowed controlling for potential effects of the moose's circadian rhythm.

To explore moose space use, we calculated diurnal activity ranges as 95% kernels (square kilometer) on the day of treatment [day 0], the 2 days before [day-2, day-1] and 1 day after treatment [day+1] using Ranges 6 v1.217 (Anatrack, Wareham, UK). We used a mixed linear model procedure with repeated statement and moose individuals as random factor using the Kenward-Roger correction to avoid type I error [START_REF] Littell | Living in snowy environments; quantifying the influence of snow on moose behavior[END_REF]). We used adjusted p values given by Tukey's post hoc test to explore the differences in diurnal activity range sizes and applied the first-order autoregressive [AR(1)] as covariance model. We evaluated the activity ranges as fixed kernels with selected cores using least squares cross-validation inflection to estimate the smoothing factor. We used an average of 54±0.3 standard error (SE) fixes per calculation. To test if moose left the area of disturbance, we calculated the overlap (in percent) of diurnal home ranges, as well as the distance (in meters) between the centroids of the diurnal ranges before and after treatment using Wilcoxon signed ranks test. Doing so, we compared overlap and distance between the diurnal ranges on the 2 days preceding treatment [day-2 to day-1] with the overlap and distance between the days preceding and following disturbance [day-1 to day+1], respectively. To avoid any dilution effect of earlier disturbance, we only used the first event of the experimental treatment in this analysis.

We used a mixed linear model to test which factors influenced moose movement rates following disturbances with the observed relative changes in movement rate after disturbance treatment as response variable. We allowed for negative estimates of covariance components using the [NOBOUND] option [START_REF] Littell | Living in snowy environments; quantifying the influence of snow on moose behavior[END_REF]). We first carried out a correlation analysis on the parameters involved using Spearman's correlation coefficient (r s ). We tested the fixed factors: (1) reproductive status; (2) air temperature; (3) wind strength; (4) precipitation; (5) disturbance distance; and (6) moose initial reaction, with the three categories representing a decreasing level of impact [fled immediately; fled after defecation; calm]; (7) snow surface hardness; (8) presence of reindeer in the area; (9) moose sinking depth in snow; and (10) activity of the moose before treatment [bedding, browsing, and walking]. At the majority of the disturbance trials, 25 out of 27 moose ranged in forested habitats. The estimated average flight distance of moose approached by backcountry skiing was 160 m±14 SE. At five out of 27 disturbance trials, we observed the focal animal when approached (average flight distance=190 m± 43 SE) and, for 15 of the remaining the 22 disturbance trials, we could estimate a flight distance indirectly due to directional changes in VHF signal (average flight distance= 149 m±12 SE). We explored the variance explained by the random effect moose individuals using variance component analysis [START_REF] Crawley | The R book[END_REF]. To reduce the number of parameters involved, we carried out a principal component analysis of the weather parameters and included principal components with eigenvalues >1. We started with a univariate model, beginning with the factors most evident from the correlation analysis and manually adding factors and their interactions to the model.

Habituation

Using a mixed linear model with repeated measures, we investigated the degree of habituation to skiing disturbance by comparing the observed relative changes in movement rate after treatment among the three different disturbance trials. Moose individuals were disturbed at different times of the day.

Energetic locomotion costs

We calculated the energy usage by first translating measures of moose chest circumference into body weight using the chest measure recorded during moose markings (390 kg±8 SE; [START_REF] Wallin | Predicting body mass from chest circumference in moose Alces alces[END_REF]. Secondly, we estimated age for each moose by evaluating tooth wear while moose were immobilized (7 years±0.5 SE; [START_REF] Ericsson | Age-specific moose Alces alces mortality in a predator-free environment: evidence for senescence in females[END_REF]. Thirdly, we used the general formula of [START_REF] Taylor | Energetics and mechanics of terrestrial locomotion[END_REF] to predict the energetic cost of terrestrial locomotion per unit body mass where we translated the relative moose response, i.e., change in movement rate (in meters per hour), into energetic cost to capture the energetic expenditure per kilogram body weight per time unit (in kilojoules per kilogram per hour). We tested differences of the calculated energy usages before and after treatment using the Wilcoxon signed ranks test. To evaluate energetic usage of moose calves accompanying adult females, we used as reference records the moose calves marked in the low alpine area in 2006 and 2007 (Ericsson, unpublished data). Moose calves were born the previous summer and would thereby have an age of 0.75 year at the timing of our disturbance. To address moose calves' energetic expenditures, we calculated their locomotion costs indirectly using average adult movement rates and calf body weight.

We used SAS 9.1.3 (SAS Institute, Cary, NC, USA) for all statistical analyses. Significance level was set to p<0.05. Data was log transformed to improve normality when needed. Average values are given with SE if not mentioned differently. We applied nonparametric tests whenever transformations failed to produce normality.

Results

Moose response was short-term, but distinct. Moose showed increased movement rates (in meters per hour) up to 3 h following disturbance (Fig. 2). During the first hour following disturbance, moose moved about 33 times as fast as they did during the corresponding hour on the previous day, coming along with an increase in moose energetic usage per kilogram body weight by 48% (before 3.6±0.1 kJ kg -1 h -1 versus after 5.4±0.5 kJ kg -1 h -1 , s=12, p=0.046). We estimated that calves that followed adult females when disturbed would face an increase in energetic expenditures of 61%. Movement rates following disturbance were correlated with snow sinking depth and with moose initial reaction (sinking depth r s =0.6, p=0.004; initial reaction r s = -0.5, p= 0.008). Individuals which increased their movement rates to the greatest extent when approached by cross-country skiers were those that responded when the skier was relatively close or those that sank deeper into snow (distance F 1,6 =9.5, p=0.02; sinking depth F 1,4 =9.1, p=0.04).

Moose showed no evidence of habituation to disturbance (p=0.7) and the variance component analysis indicated that 36% of the variance in moose response was explained by difference among individuals.

Skiing disturbance resulted in significantly enlarged diurnal activity ranges during the day of disturbance (2 days before 0.05±0.03 km 2 , 1 day before 0.07±0.04 km 2 , day of disturbance 1.35±0.44 km 2 , 1 day after disturbance 0.04± 0.02 km 2 ; F 3,22 =11.4, p=0.0001). Disturbance caused moose to relocate to a new area as shown by significantly less areal overlap after treatment versus before treatment (before 10%±5 versus after 3%±2, s=-11, p=0.03) as well as shown by the centers of moose diurnal ranges being about eight times further apart after disturbance than before disturbance with an average distance of 2,131±417 m compared to 256±68 m (s=22, p=0.008).

Discussion and conclusion

We found off-trail backcountry skiing disturbance to trigger a distinct but short-term response in moose with increased locomotion activity resulting in relocation to new area. The behavior of recreationists, predictability, frequency, and location of human activity are factors that influence the level of disturbance in wildlife [START_REF] Knight | Wildlife and recreationists: coexistence through management and research. Island, Washington Lavsund S, Sandegren F[END_REF][START_REF] Stankowich | Ungulate flight responses to human disturbance. A review and meta-analysis[END_REF]. Off-trail human activity such as backcountry skiing is less predictable for wildlife than on- trail skiing activity and thereby bear the risk to result in a larger effect zone [START_REF] Taylor | Wildlife response to recreational and associated visitor perceptions[END_REF]. Furthermore, a stimulus of unpredictable character may be more difficult to habituate to. In contrast to findings by [START_REF] Colman | Summer response distance of Svalbard reindeer Rangifer tarandus platyrhynchus to provocations by humans on foot[END_REF] for Svalbard reindeer Rangifer tarandus platyrhynchus, we did not find any evidence that moose habituated to our human-induced disturbances. We argue that the lack of habituation may be a result of the lasting human impact on moose mortality in Sweden resulting from the sustained hunting pressure in the form of high harvest rates, a long hunting season, and long period of predominance in human-related moose mortality [START_REF] Ericsson | Age-specific moose Alces alces mortality in a predator-free environment: evidence for senescence in females[END_REF]Lavsund and Sandegren 1989;[START_REF] Sand | Cross-continental differences in patterns of predation: will naïve moose in Scandinavia ever learn?[END_REF]). However, we cannot exclude that the short duration of the intervals among treatments precluded the detection of habituation, although the distinct shape of changes in moose behavior when disturbed does not give strong evidences for this alternative. We found that more than one third of the variation in moose movement rates was explained by difference among individuals. Risk assessment and degree of habituation vary among individuals in a population, and differences in behavioral responses to disturbances may be consistent for the same individuals [START_REF] Picton | Energetic cost of wildlife displacement by winter recreationists[END_REF][START_REF] Stankowich | Ungulate flight responses to human disturbance. A review and meta-analysis[END_REF]. We suggest that greater risk perception triggered stronger response as indicated by greater movement rates in individuals that responded in shorter distance to the origin of disturbance and that sank deeper in the snow. High-risk perception as potentially experienced when tracked may highlight differences among individuals to elude the area of risk.

We found that disturbance by skiing resulted in almost a doubling of expenditures per kilogram body weight for adult moose females. Because metabolic rates are affected not only by the activity itself, but also by animal's status and vary seasonally, energetic costs of disturbances need to be considered in the context of the individual's habituation status and energetic status, which may be difficult to assess in free-ranging animals [START_REF] Hudson | Maintenance metabolism[END_REF][START_REF] Picton | Energetic cost of wildlife displacement by winter recreationists[END_REF][START_REF] Schwartz | Nutrition and energetics[END_REF]. Diversity in terrain and in snow cover further complicates calculations of energy expenditures in free-ranging animals [START_REF] Bunnell | Sinking depths of black-tailed deer in snow, and their indices[END_REF]). Thus, our estimates of energy usage for increased locomotion activity, which are purely based on movement rates, reflect minimum values. Although being of shorttermed nature, such changes in energetic expenditures may be severe in areas of high human activity and for animals more sensitive in their risk perception. Moose is very well adapted to winter conditions, but especially locomotion in snow is costly and the relative cost of locomotion is higher for smaller animals as a result of their lower brisk length [START_REF] Coady | Influence of snow on behaviour of moose[END_REF][START_REF] Fancy | Incremental cost of activity[END_REF][START_REF] Littell | Living in snowy environments; quantifying the influence of snow on moose behavior[END_REF]. Moose calves may, therefore, suffer even relatively higher energy costs as our estimates when moving under deep snow conditions. Furthermore, even when utilizing its mother's tracks [START_REF] Coady | Influence of snow on behaviour of moose[END_REF][START_REF] Littell | Living in snowy environments; quantifying the influence of snow on moose behavior[END_REF], a calf may have difficulties in following when the female moves fast (as often done under risk avoidance) due to the female's greater stride length. Conclusively, moderate human disturbances of moose during wintertime may not be costly for adult moose per se if in good body condition, but may affect the body condition of accompanying subadults considerably, which, in turn, may affect population growth negatively.

Given that human-related disturbances are perceived analogously as a risk of predation, future research needs to address qualitatively and quantitatively the direct and indirect impacts of human activities on wildlife and to appropriately incorporate such knowledge into future wildlife and landscape management [START_REF] Creel | Relationships between direct predation and risk effects[END_REF][START_REF] Frid | Human-caused disturbance stimuli as a form of predation risk[END_REF][START_REF] Hebblewhite | Human activity mediates a trophic cascade by wolves[END_REF]. This is particularly needed where species are highly exposed to offtrail recreational activities and where human activities are the major source of mortality. A better understanding of the consequences is not only crucial in areas where wildlife and humans share limited space year-round [START_REF] Ingold | Freizeitaktivitäten im Lebensraum der Alpentiere[END_REF], but also for areas where disturbance frequencies peak in periods that are critical for wildlife. Popularity of different recreational activities differs among time, and because off-trail activities results in a larger area of influence than on-trail activities, research needs to follow changes in tourism attitudes [START_REF] Fredman | Changes in skiing and snowmobiling in Swedish Mountains[END_REF][START_REF] Taylor | Wildlife response to recreational and associated visitor perceptions[END_REF].

Fig. 1

 1 Fig. 1 Map of Fennoscandia with Sweden marked in gray. The study area is represented by the solid circle, representing a gradient from low alpine environments to inland boreal forest from west to east. The Arctic Circle is indicated by a line

Fig. 2

 2 Fig. 2 Change in the average rate of moose movement (in meters per hour) ± SE before and after skiing disturbance. *p<0.05, **p<0.01, ***p≤0.0001

Acknowledgements We thank the EU/Interregional IIIA project Moose in Midscandinavia, the project Moose Management in Västerbotten, the program Adaptive Management of Fish and Wildlife, the Swedish Association for Hunting and Wildlife Management, and the Swedish Environmental Protection Agency for the financial support. We are grateful to Eric Andersson and Åke Nordström for their help with the fieldwork. We thank Nils Bunnefeld for the helpful comments. The Animal Care Committee for Northern Sweden at Umeå approved the project (Dnr A124-05 2005-11-15), which was carried out in accordance with the Swedish laws concerning animal research ethics. All personnel were certified according to the standards of the Swedish Animal Welfare Agency and the Swedish Board of Agriculture.