Lionel Bringoux

Vincent Nougier
email: vincent.nougier@ujf-grenoble.fr

Pierre-Alain Barraud

Ludovic Marin

Christian Raphel

Contribution of somesthetic information to the perception of body orientation in the pitch dimension

This study investigated the contribution of otolithic and somesthetic inputs in the perception of body orientation when pitching at very slow velocities. In Experiment 1, the subjects' task was to indicate their subjective postural vertical, in two different conditions of body restriction, starting from different angles of body tilt. In the "strapped" condition, subjects were attached onto a platform by means of large straps. In the "body cast" condition, subjects were completely immobilized in a depressurized system, which attenuates gravity-based somesthetic cues. Results showed that the condition of body restriction and the initial tilt largely influenced the subjective postural vertical. In Experiment 2, subjects were displaced from a vertical position and had to detect the direction of body tilts. Results showed that the threshold for the perception of body tilt was higher when subjects were immobilized in the body cast and when they were tilted backward. Experi ment 3 replicated the same protocol from a supine starting position. Compared to results of Experiment 2, the threshold for the perception of body tilt decreased significantly. Overall, these data suggested that gravity-based somesthetic cues are more informative than otolithic cues for the perception of a quasi-static body orientation.

It is well known that sensory information from the visual, somesthetic, and vestibular systems contributes to the perceived orientation of the body in space. According to the ecological theory of orientation,severalauthors consider that the intermodal relationship between patterns of energy available to each of these sensory systems constitutes in itself high-order information that cannot be divided [START_REF] Fouque | Action and intermodal information influence the perception of orientation[END_REF][START_REF] Gibson | The senses considered as perceptual systems[END_REF]Stoffregen & Bardy,2001;Stoffregen & Riccio,1988).On the other hand, many researchers relying on sensory conflict experiments regard the perception of body orientation as based on the integration or sensory signal fusion [START_REF] Howard | Interactions within and between the spatial senses[END_REF][START_REF] Mergner | A simple model of vestibular canal-otolith signal fusion[END_REF] of gravity-based visual, vestibular, and somesthetic information. However, within this theoretical background, the respective contribution of these sensory systems-that is, the weight attributed by the central nervous system during the integration process-is still a matter of debate. As mentioned by [START_REF] Mittelstaedt | The role of otoliths in perception of the vertical and in path integration[END_REF], the perception of body orientation in total darkness is traditionally thought to be based on vestibular information and supplemented by somesthetic information. It has been proposed that otolith organs are natural graviceptive organs [START_REF] Young | Perception of the body in space: Mechanisms[END_REF] and provide accurate information about the direction of gravity forces and the orientation of the body with respect to them. As it was clearly expressed by [START_REF] Mittelstaedt | The role of otoliths in perception of the vertical and in path integration[END_REF], in the absence of all other spatial information, and under purely static conditions, the otolith system is known permanently to produce a set of afferents from which a "virtually veridical representation of the vertical" with respect to the head may be derived. Electrophysiological evidence about modifications of the discharge of primary otolith neurons during slight head tilts [START_REF] Fernandez | Physiology of peripheral neurons innervating otolith organs of the squirrel monkey. I. Response to static tilts and to long-duration centrifugal force[END_REF] contributed to convey the assumption that the otoliths signal not only the static orientation of the head to gravity, but also changes in orientation that accompany angular head displacements [START_REF] Benson | Sensory functions and limitations of the vestibular system[END_REF].

On the other hand, experiments conducted in microgravity emphasized the importance of some somesthetic cues for the perception of body orientation [START_REF] Lackner | Mutisensory, cognitive, and motor influences on human spatial orientation in weightlessness[END_REF], 2000). When subjects who where free floating without any contact with the aircraft closed their eyes, many of them lost all sense of body orientation in relation to their surroundings. For these subjects, applying pressure to the tops of their heads or under the soles of their feet made them feel upside down or rightside up, respectively. This clearly indicates that specifically oriented somesthetic stimulation is sufficient to convey information about body orientation in space. However, this does not give further knowledge about the predominance of these types of sensory cue over gravity-based otolithic information. These results simply showed that some somesthetic cues could adequately compensate for the lack of gravity-based otolithic informa tion, the latter still considered as the most accurate graviceptive organ on Earth.

Nevertheless, experiments carried out in water immersion have led to interesting results contrasting with the previous conclusion [START_REF] Brown | Orientation to the vertical during water immersion[END_REF][START_REF] Massion | Body orientation and regulation of the center of gravity during movement under water[END_REF][START_REF] Nelson | Effect of water immersion and body position upon position of gravitational vertical[END_REF][START_REF] Ross | Orientation to the vertical in free divers[END_REF]. During immersion and in a buoyant state, the somesthetic system has a limited access to gravity-based information, whereas gravity still acts on a component of the vestibular system [START_REF] Ross | Orientation and movement in divers[END_REF]. Therefore, in such an environmental condition, the specific contribution of otolithic information to the perception of body orientation in space can be more directly studied.Subjects'postural responses to the vertical under water were strikingly inaccurate, showing a forward tilt ranging from 7.3 deg [START_REF] Massion | Body orientation and regulation of the center of gravity during movement under water[END_REF] to 13.2 deg [START_REF] Ross | Orientation to the vertical in free divers[END_REF]. This suggests that gravitybased somesthetic cues may play a more important role than the otoliths for perceiving a static body orientation.

However, methodological criticisms were often associated with these experiments (e.g., Stoffregen & Riccio,1988).For instance,fixation straps or shoes [START_REF] Massion | Body orientation and regulation of the center of gravity during movement under water[END_REF][START_REF] Nelson | Effect of water immersion and body position upon position of gravitational vertical[END_REF]) may generate tactile cues that are sometimes perturbing with respect to the task requirements. When subjects were not fixed onto a platform, judgements were performed after rapid whole-body rotations [START_REF] Brown | Orientation to the vertical during water immersion[END_REF][START_REF] Ross | Orientation to the vertical in free divers[END_REF], which could lead to strong semicircular aftereffects. Overall, severalaspects can disturb subjects' perception under water, such as fluid resistance to body displacement, the relative buoyancy of the body, or the respiratory equipment [START_REF] Brown | Orientation to the vertical during water immersion[END_REF]. Therefore, other experimental methods must be used to avoid the undesirable effectsof the studies conducted in immersion aswell as in microgravity.

The present experiments were built in the frame of the previous conclusion for further investigating the respective contribution of the vestibular and somesthetic systems to quasistatic spatial orientation.More specifically,we analysed the extent to which subjects can consciously perceive their body orientation, when gravity-based tactile and proprioceptive cues are either available or attenuated by means of a specific body cast.

In Experiment 1, the subjects' task was to indicate their subjective postural vertical, starting from different angles of body tilts in a forward or backward initial position. In Experi ment 2, the subjects' task was to detect a very slow body tilt in the pitch dimension with respect to a referred vertical body orientation. In Experiment 3, instead of starting from a vertical position, subjects were initially oriented in a supine position and had to detect a tilt from this initial orientation. For each experiment, the task was executed in two conditions of body restriction. With regard to somesthetic implications enlightened in microgravity and underwater experiments, our main hypothesis postulated that errors of estimation in body orientation increased when body restriction increased, because oftactile and proprioceptive alteration.Moreover,it was expected that the direction of tilt, as wellas the initialbody orientation, might lead to differences in spatial orientation judgements, mainly because of interoceptive influences [START_REF] Mittelstaedt | Somatic versus vestibular gravity reception in man[END_REF][START_REF] Mittelstaedt | The role of otoliths in perception of the vertical and in path integration[END_REF].

GENERAL METHOD

In all experiments reported here, subjects were restrained on a servo-controlled motorized platform (Figure 1) allowing backward and forward rotations in the sagittal plane (pitch dimension) at a constant velocity of 0.05 deg s, following an initial acceleration phase at 0.005 deg s 2 . Moving the platform at this very slow velocity prevented responses of the semicircular canals [START_REF] Goldberg | The vestibular system[END_REF]. Position signals from the platform were sampled at 20 Hz (12-bit A/D converter). Earphones providing white noise masked the noise induced by the motor during rotation.

Two conditions of body restriction were manipulated (strapped and body cast). In the socalled strapped condition (Figure 1), subjects were attached onto the platform by means of six large straps fixed at the level of the head, shoulders, chest, hips, knees, and ankles. In the body cast condition, subjects were completely immobilized in a specific device looking like a body cast. This body cast was composed of three plastic envelopes full of polystyrene micromarbles and connected to a depressurization device. The surface of the plastic envelopes was very thin in order to ensure the closest contact possible with the subjects' body. The wrapping was done in the supine posture with no contact of the soles with the foot-rest. The back envelope was directly attached onto the platform and entirely covered the back of the subjects' body from the head to the feet. The front envelope was put onto the front of the subjects' body from the neck to the feet and was maintained by means of the straps. The third envelope specifically immobilized the head and shoulders. When subjects were appropriately wrapped, the body cast was depressurized.

A preliminary experiment was conducted to investigate the capability of the body cast to minimize the gravity-based tactile stimulation-that is, to create a more diffuse and isotropic pattern of pressure around the body and to suppress important changes in pressure repartition that occur for different body tilts.

Two subjects (one male, 1.74 m, 76 kg ; one female, 1.62 m, 58 kg) were restrained on the movable platform in the two strapped and body cast conditions. Subjects were tilted at pitch angles ranging from -90 deg to +90 deg by steps of 15 deg. Pressure variations were recorded at six different parts of the body by means of force sensing resistors. The variation of resistance as a function of force gives an estimation of the variation of pressure generated at the level of the captor, with the output resistance increasing as the force decreases. The six resistor loca tions were distributed as follows: three on the posterior part of the body (D-10 vertebra, ischiatic tuberosity, and left gastrocnemius proximal insertion), two on the anterior part of the body (substernal central point and patella base), and one under the feet (first phalanx of the left big toe).

As illustrated in Figure 2, results obtained from the two subjects did not differ significantly. This observation showed that the different morphologicalcharacteristics of the subjects do not interact with the distribution and evolution of pressure cues with respect to body orientation. Regarding the variations of resistance measured by the force-sensing resistors at the six body locations, there was no difference between the two conditions of body restriction for two locations only:the substernal centralpoint and the patella base. This fact can be easily explained considering that the force-sensing resistors placed at the anterior part of the body in the strapped condition were fixed just under the tightened straps. To some extent, it recreated locally the body cast condition.

Under the sole of the feet (first phalanx of the left big toe), resistance values differed for extreme body tilts only (> ±60 deg). When the subjects were tilted over 60 deg towards the horizontal supine or prone body orientation, a take off occurred in the strapped con dition, leading to no contact,while pressure remained nearly constant in the body cast condition.

Regarding the posterior part of the body, the resistance level was almost identical for both conditions of body restriction for the backward angles of tilt. However, in the strapped condition, the resistance values increased abruptly near the vertical to reach infinity for the forward tilt angles.Conversely,in the body cast condition, the resistance values still remained nearly constant or slowly increased when the body was tilted forward, over the vertical. These data clearly showed that the posterior parts of the body took off from the platform in the strapped condition, unlike in the body cast condition. This event occurred at different angles of tilt around the vertical, according to each body location. It began by the left gastrocnemius proximal insertion at approximately -15 deg backwards followed by the D-10 vertebra just around the vertical, and then by the ischiatic tuberosity around +30 deg forwards.

To summarize, in the strapped condition, slight tilts led to a consistent modification in the repartition of pressure exerted on the body by the support surface. Gravity-based tactile and proprioceptive cues were then still clearly present. In the body cast condition, however, pressure generated by antigravity reaction forces was still present but drowned in a wholeisotropic pattern of pressure stimulation, which was nearly invariant whatever the body tilt. Hence, if the body cast condition could not obviously be assimilated to a tactile deprivation situation,the gravity-based tactile cues were nevertheless less discernable for the central nervous system. Moreover, because of the suppression of postural activity, whatever the body orientation, gravity cues sensed by "load" receptors were no more available [START_REF] Dietz | Regulation of bipedal stance: Dependence on "load" receptors[END_REF]. Consequently, compared to the strapped condition, it can be stated that gravity-based somesthetic cues were attenuated in the body cast.

EXPERIMENT 1

The purpose of Experiment 1 was to investigate the respective vestibular and somesthetic contribution to the judgement of the subjective postural vertical. In this classical task [START_REF] Bisdorff | The perception of body verticality (subjective postural vertical) in peripheral and central vestibular disorders[END_REF]Clark & Graybiel,1963), subjects were asked to estimate their perceived verticality, starting from various tilted posi tions in the pitch dimension.

BRINGOUX ET AL.

Bronstein

Methods

Subjects

A total of 10 subjects (7 males and 3 females, mean age = 27 years) participated in Experiment 1. All subjects gave signed informed consent in compliance with the ethical committee (i.e., Helsinki Conven tion), which governs and regulates human experimentation in France. As in the following experiments, none of these subjects reported any sensation of discomfort, which could interfere with their judgement skills.

Procedure

The subjects' task was to verbally indicate when they felt vertical in the pitch dimension in total dark ness. The two conditions of body restriction presented above were investigated in two counterbalanced sessions. Each session was composed of six trials crossing randomly two directions of initial orientation (forward and backward) × three angles of initial orientation (10, 20, and 30 deg). Subjects were first tilted to the desired initial orientation at a constant velocity of 4 deg s. After 20 s, allowing the information issued from the semicircular canals to be close to zero [START_REF] Goldberg | The vestibular system[END_REF], the tilting platform started its slow displacement (0.05 deg s) back to the direction of the gravitational vertical. Subjects were encouraged to give their response with a confidence level of minimum 4 on a 5-point scale. At the end of each trial-that is, after the subjective postural vertical was judged, subjects were placed in the upright position at a random velocity, to avoid any estimation of the performance, and the lights turned on for 30 s, to avoid any postural after effect or disorientation illusion prior to the next trial. No verbal feedback about performance was given at any time. Furthermore, subjects were not instructed either about the direction and magnitude of the initial orientation, or about the angular velocities of platform rotation. For making their judgement, subjects were strongly encouraged to use available body sensations rather than making inferences about the time they spent during one trial.

Results and discussion

The mean absolute error of judgement with respect to gravity was used to express the subjec tive postural vertical, independently of the signed direction of the body tilt. A 2 (conditions of body restriction: strapped and body cast) × 2 (directions of initial tilt: forward and backward) × 3 (angles of initial tilt: 10, 20, and 30 deg) analysis of variance (ANOVA) with repeated measures on the three factors was applied to the data. Results showed that the mean error of judgement was smaller in the strapped than in the body cast condition (4.19 deg ± 3.10 vs. 6.49 deg ± 4.86, respectively), F(1, 9) = 9.94, p < .05, accounting for 20.3% of the variance. There was also a main effect of the angle of initial tilt, F(2, 18) = 25.87, p < .001, accounting for 57.4% of the variance, but no effect of the direction of this initial tilt. A post hoc analysis (Scheffé test) showed that the error of judgement increased from 10 to 30 deg (3.1 deg ± 1.0, 5.1 deg ± 2.3, and 7.9 deg ± 3.0, for the 10-, 20-and 30-deg tilted positions, respectively, ps < .05). Observa tion of the data showed that the errors of judgement consistently deviated in the direction of the initial tilt. This effect increased when increasing the angle of tilt. Finally, there was also a significant interaction of body restriction by angle of initial tilt, F(2, 18) = 6.66, p < .01. As illustrated in Figure 3, the effect of the angle of initial tilt was greater in the body cast than in the strapped condition.

It is well known that both normal and labyrinthine defective subjects are able to give fairly accurate estimates of the subjective postural vertical, with errors smaller than 2 PERCEPTION OF BODY ORIENTATION degrees, when gravity-based somesthetic cues are still available [START_REF] Anastasopoulos | Dissociation between the perception of body verticality and the visual vertical in acute peripheral vestibular disorder in humans[END_REF][START_REF] Bisdorff | The perception of body verticality (subjective postural vertical) in peripheral and central vestibular disorders[END_REF][START_REF] Clark | Perception of the postural vertical in normals and subjects with labyrinthine defects[END_REF]. The present results clearly suggested that the somesthetic system plays a major role in estimating the direction of subjective postural vertical [START_REF] Bronstein | The interaction of otolith and proprioceptive information in the perception of verticality. The effects of labyrinthine and CNS disease[END_REF]. The greater the limitation of the contribution of the somesthetic system, the greater was the error of judgement with respect to the gravitational vertical and to the initial angle of tilt. This also indicates that the otoliths are unable to compensate for the lack of gravity-based tactile and proprioceptive information.

In a task involving subjective postural vertical judgements, subjects had to associate a specific multisensory configuration to a particular postural orientation. Previous authors proposed that the central nervous system incorporates information about body dynamics and sensory dynamics to develop an internal model [START_REF] Lestienne | Postural control in weightlessness: A dual process underlying adaptation to an unusual environment[END_REF][START_REF] Merfeld | A multidimensional model of the effect of gravity on the spatial orientation of the monkey[END_REF][START_REF] Merfeld | Humans use internal models to estimate gravity and linear acceleration[END_REF]. The output of this internal model-thatis, expected sensory afferences for a particular body position-is compared to the actual available sensory afferences.The error between this perceptual internal reference developed through subjects'experience [START_REF] Bringoux | Effects of gymnastics expertise on the perception of body orientation in the pitch dimension[END_REF] and the actual sensory afferences would be used to drive the central estimate towards the value of reference. In the absence of enough somesthetic information about body orientation with respectto their own reference of verticality [START_REF] Lestienne | Postural control in weightlessness: A dual process underlying adaptation to an unusual environment[END_REF], the subjects tended to perceive their current body position as nearer to the upright reference position than it was in reality.

The same argument can explain the differences observed with respect to the angle of initial tilt. Because of the very slow rotation of the platform, changes in the somesthetic cues over time are very low leading to an underestimation of the current body tilt [START_REF] Higashiyama | Apparent body tilt and postural aftereffect[END_REF]. As previously indicated, this underestimation increased in the body cast condition (Figure 3).

EXPERIMENT 2

The purpose of Experiment 2 was to investigate the respective contribution of gravity-based somesthetic and vestibular cues in the perception of a slow body tilt from an initial vertical body orientation. This task can be considered to some extent as the reverse of the previous subjective postural vertical task, subjects having to perceive a tilt from a vertical position instead of having to perceive the vertical from a tilted position. Threshold for the perception of a change in body orientation was defined for both conditions of body restriction and both directions of body tilt.

Method

Subjects

A total of 8 new subjects (5 males and 3 females, mean age = 26 years) participated in the study. All subjects gave informed consent and were naive about the purpose of the experiment.

Task and procedure

The subjects' task was to perceive backward and forward slow body tilts in total darkness, starting from an initial vertical body orientation. Prior to each trial, subjects were oriented in the initial position. When ready, the trial was initiated, and it ended with the detection of a body tilt. It has been emphasized elsewhere [START_REF] Fitzpatrick | Proprioceptive, visual, and vestibular threshold for the perception of sway during standing in humans[END_REF][START_REF] Teasdale | Contribution of ankle, knee and hip joints to the perception threshold for support surface rotation[END_REF] that the detection of a body tilt can occur before the direction of the pitch can be reported. Subjects were instructed to indicate verbally when they perceived a change in body orientation and the direction of tilt. In order to obtain responses that exceeded that of simple chance, subjects were also instructed to give their level of confidence on a 5point scale. They were encouraged to postpone their response until reaching Level 4 on the 5-point scale. Following the response, the platform was brought back to the starting position at a random velocity to avoid any estimation of the performance. Furthermore, no verbal feedback about subjects' performance was given at any time. The two conditions of body restriction were tested in two separate blocks of trials presented randomly. For each condition, 4 trials for each pitch direction (backward and forward) and 2 catch trials with the platform remaining immobile were executed randomly for a total of 10 trials. The catch trials were used to further ensure that subjects did not detect movements of the platform by guessing. The duration of these trials varied randomly from 120 to 300 s, prior to indicating to the subjects that a new trial would be initiated. When an error of judgement was made, an additional identical trial was randomly inserted in the block. Therefore, subjects were not told either about the number of trials that composed a block, or about the direction of tilt.

Results and discussion

Errors of judgement. Overall, subjects made judgement errors in 10.5% and 24.5% of the trials in the strapped and body cast conditions, respectively. Among these errors, 30% were due to a wrong perception of the direction of tilt during an effective platform rotation; the 70% remaining errors were due to a wrong perception of body tilt during catch trials. In all cases, the erroneous reported direction of tilt was different across trials and subjects. Detection thresholds. A 2 (body restriction: strapped, body cast) × (2 pitch direction: back ward, forward) ANOVA with repeated measures on all factors was applied to the threshold for the perception of a body tilt. Results showed main effects of body restriction, F(1, 7) = 80.12, p < .001, accounting for 55.1% of the variance, and direction, F(1, 7) = 51.95, p < .001, accounting for 37.5% of the variance. Threshold for the perception of a body tilt was lower in the strapped than in the body cast condition (7.6 deg ± 1.6 vs. 15.8 deg ± 1.6, respectively). It was also lower when the platform pitched forward than when it pitched backward (8.3 deg ± 1.5 vs. 15.1 deg ± 1.7, respectively). Results also showed a significant two-way interaction of body restriction by direction, F(1, 7) = 13.94, p < .01. As illustrated in Figure 4, the effect of restriction was greater when pitching backward than when pitched forward.

Results of the present experiment showed that the threshold for the perception of a body tilt was higher when subjects were completely immobilized in the body cast than when partially restrained. Once again, gravity-based tactile and proprioceptive sensory cues seemed to play a major role in the perception of body orientation. The very high threshold observed in the body cast condition supported the results of various authors [START_REF] Fouque | Action and intermodal information influence the perception of orientation[END_REF][START_REF] Riccio | The role of balance dynamics in the active perception of orientation[END_REF][START_REF] Teasdale | Contribution of ankle, knee and hip joints to the perception threshold for support surface rotation[END_REF]. These authors suggested that the percep tion of body orientation is disturbed when proprioceptive information is not dynamically inte grated, and that otolith organs cannot naturally convey absolute graviceptive information at a perceptual level in quasi-static conditions.

Results also showed that the threshold for the perception of a body tilt was dependent on the direction of tilt. It may be hypothesized that another part of somesthetic information is differentially involved from one direction to another. Recent experimental works have shown that information provided by internal organs could play a role in postural orientation [START_REF] Von Gierke | Differences in otolith and abdominal visceria graviceptor dynamics: Implications for motion sickness and perceived body position[END_REF][START_REF] Mittelstaedt | Somatic versus vestibular gravity reception in man[END_REF][START_REF] Mittelstaedt | The role of otoliths in perception of the vertical and in path integration[END_REF][START_REF] Mittelstaedt | The relative effect of saccular and somatosensory information on spatial orientation and control[END_REF]. Interoceptive cues could probably account for such a directional asymmetry already found by [START_REF] Nemire | Visual and somesthetic influences on postural orientation in the median plane[END_REF]. The following experiment was carried out to further explore the potential role of interoception in the perception of a change in body orientation. To study the potential interoceptive components of somesthetic information in the perception of body tilt, we replicated Experiment 2, with subjects oriented in a supine starting position. It was expected that perception of body orientation also may be dependent on the hydrostatics of the blood [START_REF] Mittelstaedt | Somatic versus vestibular gravity reception in man[END_REF]-that is, subjects made fewer errors when the pitch rotation induced a body orientation with the head toward the bottom than when the feet were toward the bottom.

Method

Subjects

A total of 8 subjects (4 females and 4 males, mean age 26.2 years) participated in Experiment 3. Of these, 6 were new recruited subjects whereas 2 also took part in Experiment 2. They all gave informed consent and were naive as to the purpose of the experiment.

Task and procedure

The procedure was the same as that in Experiment 2, except that the subjects had to detect a slow change in body orientation from an initial supine position.

Results and discussion

Errors of judgement. Overall, subjects made judgement errors in 4.5% and 10.5% of the trials in the strapped and body cast conditions, respectively. Among these errors, 30% were due to a wrong perception of the direction of tilt during an effective platform rotation; the 70% remaining errors were due to a wrong perception of body tilt during catch trials. In all cases, as in the previous experiment, the erroneous reported direction of tilt was different across trials and subjects. Detection thresholds. A 2 (body restriction: strapped, body cast) × 2 (pitch direction: back ward, forward) ANOVA with repeated measures on all factors was applied to the threshold for the perception of a body tilt. Results showed main effects of body restriction, F(1, 7) = 10.87, p < .05, accounting for 56.1% of the variance, and direction, F(1, 7) = 6.13, p < .05, accounting for 35.4% of the variance. Figure 5 illustrates the results obtained in Experiment 3.

Although less consistent than in Experiment 2, the effect of body restriction led to a threshold for the perception of a body tilt that was lower in the strapped than in the body cast condition (5.2 deg ± 1.6 vs. 7.8 deg ± 2.1, respectively). However, contrary to Experiment 2, the threshold was higher when the platform pitched forward than when it pitched backward (7.6 deg ± 2.6 vs. 5.5 deg ± 1.0, respectively). Furthermore, observation of the data showed that the thresholds observed in Experiment 3 were lower than those in Experiment 2. These results strongly suggested that a particular source of information is better used starting from a supine position. As proposed by [START_REF] Mittelstaedt | Somatic versus vestibular gravity reception in man[END_REF], influence of gravity on the cardiovascular system should be a good candidate. Variations of homeostatic pressure with body tilt around the horizontal position appear as very informative cues, which can compensate for the tactile and proprioceptive disturbances generated by the body cast. Moreover, when subjects were tilted backward, such an unusual and alarming situation probably lowered the threshold for the perception of a body tilt, mainly because of the rush of blood to the head.

GENERAL DISCUSSION

Results of the present experiments showed that the perception of body orientation was more accurate when subjects were partially restrained than when they were completely immobilized in the body cast. In that condition, the appropriate use of gravity-based tactile and proprioceptive information was altered. The overall pattern of somesthetic stimulation gener ated by antigravity reaction forces thus appears to be highly informative for the perception of body orientation [START_REF] Lackner | Mutisensory, cognitive, and motor influences on human spatial orientation in weightlessness[END_REF], 2000).

As mentioned by [START_REF] Higashiyama | Apparent body tilt and postural aftereffect[END_REF], tactile mechanoreceptors, distributed over the wide area of cutaneous tissue, appear to provide the central nervous system with spatially and temporally coded information associated with changes in the distribution of pressure on the skin that result from changes in body orientation. Thus, the important contri bution of tactile mechanoreceptors to perceived body orientation suggested by our results might greatly depend on the availability of changing pressure patterns generated during body rotation [START_REF] Bringoux | Perception of slow pitch and roll body tilts in bilateral labyrinthine-defective subjects[END_REF][START_REF] Lackner | Some influences of touch and pressure cues on human spatial orientation[END_REF]. Similarly, changes in muscular tension opposed to the attraction of gravity, which are eliminated in the body cast, may play a major role in perceiving a body tilt. In addi tion, "load receptors" such as Golgi tendon organs have been shown to yield gravity-based information during active postural stance [START_REF] Dietz | Regulation of bipedal stance: Dependence on "load" receptors[END_REF]. Thus, antigravity muscular activity might also generate cues at a perceptual level, informing the subjects about their orien tation in space. The present results also showed that when the use of visual, vestibular, tactile, and proprioceptive information is altered, sensory inputs from truncal graviceptors [START_REF] Mittelstaedt | Somatic versus vestibular gravity reception in man[END_REF][START_REF] Mittelstaedt | Interaction of eye-, head-, and trunk-bound information in spatial perception and control[END_REF][START_REF] Mittelstaedt | The relative effect of saccular and somatosensory information on spatial orientation and control[END_REF] perceive body orientation in space. Mittelstaedt and collaborators, on the basis of "subjective horizontal position" experiments conducted with paraplegics and nephrectomized patients on a tiltable board and on a sled centrifuge, enlightened at least two separate truncal graviceptive inputs. A first input could originate in renal afferences about pressure exerted towards the surface of the kidney. A second input has been shown to yield gravity information through the inertia of a mass in the body. The mass of blood in the large vessels, the mass of the abdominal viscera [START_REF] Von Gierke | Differences in otolith and abdominal visceria graviceptor dynamics: Implications for motion sickness and perceived body position[END_REF], and hydrostatic effects have been respectively evoked as potential sources for this second input. Lower tilt detection threshold in supine than in vertical initial orientation, as well as the reversed effect of the direction of tilt, might be due to an increased relevance of some of the latter sources of interoceptive information. In the supine position, for example (Experiment 3), the homeostatic pressure was exerted in a reversed Zaxis direction when pitching backward and forward. When pitching backward, the homeostatic pressure increased significantly at the head level, allowing a fast detection of a change in body orientation. When pitching forward, the variation of hydrostatic pressure at the level of the feet was something natural and less significant for the subjects, as the reference position for the human being is the bipedal stance. In that position (Experiment 2), the varia tion of homeostatic pressure probably did not contribute to the detection of the body tilt because these variations are similar when pitching forward and backward. Other interoceptive signals may have been differently processed by the central nervous system such as the mass of the viscera [START_REF] Von Gierke | Differences in otolith and abdominal visceria graviceptor dynamics: Implications for motion sickness and perceived body position[END_REF]Mittelstaedt,1992[START_REF] Mittelstaedt | Interaction of eye-, head-, and trunk-bound information in spatial perception and control[END_REF][START_REF] Mittelstaedt | The relative effect of saccular and somatosensory information on spatial orientation and control[END_REF], depending on initial body orientation as well as on tilt direction. Following this hypothesis, movements of the viscera would be greater when pitching forward than when pitching backward (Experiment 2). This would result in a lower threshold for the perception of a body tilt. Further experiments(e.g., with paraplegics)need to be carried out to test this interpretation.

These results also indicated that, under specific constraints such as for a complete static posture, the availability of otolith inputs does not imply a better perception of body orientation. From an electrophysiological point of view, it is known that slight modifications of head orientation lead to modulations of the otolith afferents discharge [START_REF] Fernandez | Physiology of peripheral neurons innervating otolith organs of the squirrel monkey. I. Response to static tilts and to long-duration centrifugal force[END_REF]. In the present experiment, however, when subjects were tilted at 0.05 deg s, it may be advanced that the speed of variation of the otolithic signal through time was not sufficiently high to have a significant effect on behaviour. Overall, these findings suggest that, at a percep tual level, the otolith organs cannot be considered as efficient graviceptors in quasi-static conditions [START_REF] Teasdale | Contribution of ankle, knee and hip joints to the perception threshold for support surface rotation[END_REF]. The vestibular inputs may become much more useful only when the signal to noise ratio reaches an unambiguous level, such as for more dynamical situa tions (e.g., for more rapid tilts), or with trained subjects, such as gymnasts [START_REF] Bringoux | Effects of gymnastics expertise on the perception of body orientation in the pitch dimension[END_REF].

From another point of view, these results also support those of various authors [START_REF] Fouque | Action and intermodal information influence the perception of orientation[END_REF][START_REF] Riccio | The role of balance dynamics in the active perception of orientation[END_REF][START_REF] Teasdale | Contribution of ankle, knee and hip joints to the perception threshold for support surface rotation[END_REF] showing that the disturbance of dynamic information altered perceived orientation. The ecological theory of orientation suggested that a correct perception of the self-orientation in space requires a dynamic processing of all the available sensory information [START_REF] Stoffregen | An ecological theory of orientation and the vestibular system[END_REF]. In the body cast condition, subjects were completely restrained so that they had no active control of any part of their body (except the eyes).By contrast,the level of restraint was reduced in the strapped condition, in which active postural control was not eliminated. It is then likely that the difference in "controllability" between the two conditions of body restriction could account for the differ ence observed in the perceptual responses. According to [START_REF] Stoffregen | An ecological theory of orientation and the vestibular system[END_REF], a higher level of dynamic control of posture generates information that is not available to passive observers. Within the central nervous system, the different sensory inputs have to interact with each other and to be linked to subject's activity, in order to have a significant effect on behaviour. Recent neurophysiological studies support this claim, showing that the activation of vestibular nucleus neurons in response to tilt is not exclusively the result of labyrinthine inputs, but also of limb and trunk afferences [START_REF] Yates | Responses of vestibular nucleus neurons to tilt following chronic bilateral removal of vestibular inputs[END_REF] as well as efferent signals for postural control [START_REF] Matsuyama | Vestibulospinal and reticulospinal neuronal activity during locomotion in the intact cat. I. Walking on a level surface[END_REF].

In conclusion, the most striking result of the present experiments was that, for very slow velocities, the perception of body orientation was dependent on the characteristics of the avail able sensory inputs with respect to body restriction and initial body position. Contrary to the common assumption, signals issued from the vestibular system were not naturally efficient in quasi-static conditions for accurately perceiving the body orientation with respect to the gravity field. On the other hand, the presentresults confirmed the prominentrole of somesthetic information in postural orientation, such as tactile, proprioceptive [START_REF] Higashiyama | Apparent body tilt and postural aftereffect[END_REF], and interoceptive [START_REF] Mittelstaedt | Somatic versus vestibular gravity reception in man[END_REF] cues.

Figure 1 .Figure 2 .

 12 Figure1. Illustration of the experimental set-up. The motorized platform rotated around the central axis. In the strapped condition (left), subjects were attached onto the platform by mean of six large straps fixed at the level of the head, shoulders, chest, hips, knees, and ankles. In the body cast condition (right), subjects were completely immobilized in a depressurized body cast fixed onto the platform.

Figure 3 .

 3 Figure 3. Mean error of subjective postural vertical judgement with respect to gravity and standard deviation for the two conditions of body restriction and the three angles of initial body tilt (Experiment 1).

Figure 4 .

 4 Figure 4. Mean angular threshold and standard deviation for the perception of a body tilt starting from a vertical position for the two conditions of body restriction and the two directions of tilt (Experiment 2).

Figure 5 .

 5 Figure5. Mean angular threshold and standard deviation for the perception of a body tilt starting from a supine position for the two conditions of body restriction and the two directions of tilt (Experiment 3).

We are grateful to the Instrumentation and Technological Research Service of the Centre


Claire Lemaire
email: lemaire@cgm.cnrs-gif.fr

Patrice Hamel

Jean Velours

Geneviève Dujardin

Jean Veloursʈ

Genevie ` Ve Dujardin

Absence of the Mitochondrial AAA Protease Yme1p Restores F_0-ATPase Subunit Accumulation in an oxa1 Deletion Mutant of Saccharomyces cerevisiae

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in ⌬oxa1, ⌬oxa1⌬afg3, and ⌬oxa1⌬yme1 mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F 0 subunits Atp4p, Atp6p, and Atp17p are stabilized in the ⌬oxa1⌬yme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.

In mitochondria, biogenesis of the respiratory complexes requires the expression of both the mitochondrial and nuclear genomes (1)(2)(3). The mitochondrial genome encodes only a few subunits of the respiratory complexes, whereas the other subunits and a number of proteins, which are not intrinsic components of the complexes but are required for their biogenesis, are nuclearly encoded.

The Saccharomyces cerevisiae nuclear gene OXA1 encodes such an assembly-assisting factor that is required for the biogenesis of both cytochrome c oxidase (Cox) 1 and ATPase (4,5).

Oxa1p presents five hydrophobic segments and is located within the mitochondrial inner membrane (6 -8). In the absence of Oxa1p, oligomycin-sensitive ATPase activity is significantly decreased, and Cox activity is totally abolished. On non-denaturing gels, the Cox complex of ⌬oxa1 strains displays a higher mobility probably due to the lack of accumulation of the mitochondrially encoded subunits (9,10). In addition, OXA1 inactivation prevents the N-terminal maturation of the precursor of Cox2p (pre-Cox2p) (11) and affects its translocation (export) from the matrix to the intermembrane space (12,13). Defective insertion of several chimeric proteins in the inner membrane has also been observed in oxa1 mutants, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery [START_REF] Hell | Proc. Natl. Acad. Sci. U. S. A[END_REF]. However, the fact that the absence of Oxa1p is compensated by point mutations in the cytochrome c 1 gene is difficult to conciliate with a unique role for Oxa1p as a mitochondrial channel (10).

Two mitochondrial proteases (Afg3p-Rca1p and Yme1p) are also involved in respiratory complex assembly in yeast [START_REF] Thorsness | [END_REF](16)(17)(18). The hetero-oligomeric complex Afg3p-Rca1p acts on the matrix side of the inner membrane, whereas Yme1p is active in the intermembrane space (19,20). Afg3p-Rca1p appears to be involved in the degradation of Cox and ATPase subunits (21), and Yme1p in the degradation of Cox2p (22)(23)(24). In addition to their proteolytic activity, these proteases seem to display a chaperone-like activity (25)(26)(27)(28), and it has been shown that the overexpression of OXA1 can partially compensate for the inactivation of AFG3 (29).

Finally, Oxa1p as well as both protease complexes appear to be conserved through evolution, showing the importance of their function. The human, Arabidopsis, and Schizosaccharomyces pombe OXA1 genes have been identified using functional complementation of a yeast oxa1 mutation (30 -32). Human cDNAs encoding proteins highly related to Afg3p-Rca1p have also been described, and one of them encodes paraplegin, which, when mutated, is responsible for a hereditary spastic paraplegia (33,34).

In this work, we have studied the relationships between Oxa1p and the two inner membrane AAA (ATPases associated with diverse cellular activities) proteases by analyzing the assembly and activity of the Cox and ATPase complexes in ⌬oxa1, ⌬oxa1⌬afg3, and ⌬oxa1⌬yme1 mutants. We show that membrane subunits of both complexes, whether mitochondrially or nuclearly encoded, are rapidly degraded in the absence of Oxa1p. Interestingly, neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits, whereas F 0 -ATPase subunits are stabilized in the absence of Yme1p. The increased stability of the ATPase subunits in the ⌬oxa1⌬yme1 double mutant allows significant assembly to occur since oligomycin-sensitive ATPase activity is restored. Altogether, our results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in S. cerevisiae, we show that the simultaneous inactivation of the AFG3 and YME1 genes is lethal.

EXPERIMENTAL PROCEDURES

Strains and Plasmids-Yeast genetic methods were previously described (35). All strains are isonuclear to the wild-type strain CW04 (␣ ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100), except for the mating type, OXA1, AFG3, and YME1 loci. CW04 and NBT1 (␣ oxa1::LEU2) were previously described (4). AFG3-2 (a afg3::TRP1) exhibits the Yepafg3-2 plasmid carrying the URA3 gene and the afg3-E559Q allele (21,26). PHT7 (␣ yme1::URA3) and PHT9 (␣ yme1::Kan R ) were constructed by inactivating the YME1 gene using a URA3 cassette [START_REF] Thorsness | [END_REF] or a Kan R cassette (36). F01-6B (␣ afg3::HIS3) carries the afg3::HIS3 inactivation (25). The four double mutants were constructed either by gene inactivation (PHT8, ␣ oxa1::LEU2 yme1::URA3) or by crosses, sporulation, and microdissection (F01-6C, a oxa1::LEU2 afg3::HIS3; RF0-7C, ␣ oxa1::LEU2 afg3::HIS3; and YA1-2C, ␣ afg3::TRP1 yme1:: Kan R ). Strains RF0-7C and YA1-2C carry the Yepafg3-2 plasmid.

Purification and Extraction of Mitochondria-Purification of mitochondria and carbonate extraction were performed as described (6), except that the following protease inhibitors were added to each buffer: phenylmethylsulfonyl fluoride (1 mM), pepstatin (1 g/ml), chymostatin (10 g/ml), antipain (10 g/ml), and leupeptin (10 g/ml). The mitochondrial protein concentration was determined using the Bio-Rad assay.

In Vivo Labeling of the Mitochondrial Translation Products and Whole Cell Extracts-Yeast cells were grown on medium containing 1% yeast extract, 1% Bacto-peptone, 2% galactose, 0.1% glucose, and 20 g/ml adenine and harvested at mid-exponential phase. In vivo labeling of the mitochondrial translation products was performed essentially as described (37). Cells (1.8 ϫ 10 8 ) were harvested and resuspended in labeling medium (40 mM K 2 HPO 4 (pH 7.4) and 2% galactose). They were labeled for 20 min at 30 °C with [ 35 S]methionine (130 Ci/ml) in the presence of cycloheximide (150 g/ml) to inhibit cytosolic protein synthesis. The reaction was stopped by addition of methionine (10 mM final concentration). Total cell proteins were extracted by alkaline lysis (38).

Extraction of the F 1 Sector and F 0 Subunits-F 1 was released from mitochondrial membranes as described previously (39), except that protease inhibitors were added to the extraction buffer as in the carbonate extraction medium (see above). Mitochondria were suspended to a concentration of 5 mg/ml in extraction buffer (0.25 M sucrose, 10 mM Tris-HCl, and 1 mM EDTA (pH 7.5)), and 0.5 volume of chloroform was added. The two phases were vigorously mixed for 30 s and then separated by centrifugation at 400 ϫ g for 5 min at 20 °C. The aqueous layer was centrifuged at 100,000 ϫ g for 30 min at 20 °C to recover the supernatant, which contained the F 1 sector. F 1 -ATPase activity was then measured as described (10). For gel electrophoresis analysis, F 1 was precipitated with (NH 4 ) 2 SO 4 added to 60% saturation. Atp9p was extracted by organic solvents (40), except that mitochondrial protein (2 mg) was incubated for 2 h at room temperature with chloroform/methanol (1:1).

Cytochrome c Oxidase and ATPase Activity Measurements on Purified Mitochondria-Cox activity was estimated by measurement of cytochrome c oxidation performed spectrophotometrically at 550 nm (10). ATPase activity was measured by colorimetric determination of inorganic phosphate released from ATP (41).

Electrophoresis and Immunoblotting-Electrophoresis and electrophoretic transfers were performed as described previously (10), except for the analysis of the mitochondrial translation products (37). Immunodetection was carried out using the enhanced chemiluminescence method (Pierce). The anti-yeast Cox1p, Cox2p, Cox3p, and Cox4p monoclonal antibodies were purchased from Molecular Probes, Inc., whereas the anti-yeast Cox5p and Cox6ap monoclonal antibodies were a generous gift from R. A. Capaldi (University of Oregon) (42). The anti-yeast Cox6p polyclonal antibody was kindly provided by R. O. Poyton (University of Colorado). The anti-yeast ATPase subunit polyclonal antibodies were prepared against either the purified subunits (Atp1p, Atp2p, Atp3p, Atp4p, Atp5p, Atp16p, and Atp17p) or a synthetic peptide of the N-terminal part (Atp6p).

RESULTS

Oxa1p Is Required for the Accumulation of the Membrane Subunits of Cox and ATPase Complexes-To investigate the subunit composition of the Cox and ATPase complexes in the absence of Oxa1p, mitochondria of the wild-type and oxa1deleted (⌬oxa1) strains were treated with carbonate, and the steady-state levels of the main subunits of these two complexes were systematically analyzed in the soluble and membrane fractions by immunoblotting. For the ATPase complex, the two sectors F 1 and F 0 were also separately extracted using different organic solvents, and the subunits were revealed by immunoblotting or silver staining.

As previously shown (9, 10), the two membrane subunits Cox1p and Cox2p were not detectable in the absence of Oxa1p (Fig. 1A). The three other membrane Cox subunits were also either not detectable (Cox6ap) or poorly detectable (Cox3p and Cox5p) in the ⌬oxa1 mutant. The two subunits Cox4p and Cox6p, distributed between the membrane and the soluble fractions in the wild-type strain, were present mostly in the soluble fraction in the absence of Oxa1p, and their steady-state level was decreased.

The ATPase complex consists of a soluble sector (F 1 ) and a membrane-anchored sector (F 0 ) that assembles independently. The ATPase activity of the chloroform-extracted F 1 sector was lowered ϳ2-fold in the ⌬oxa1 strain compared with the wildtype strain (4815 Ϯ 30 versus 9870 Ϯ 450 nmol of ATP hydrolyzed per min/mg of protein). As shown by immunoblotting, the four F 1 subunits Atp1p, Atp2p, Atp3p, and Atp16p and the subunit Atp5p (oligomycin sensitivity-conferring protein subunit) were decreased by ϳ40% in the ⌬oxa1 extracts (Figs. 2A and3A), which is consistent with the activity measurement. On the contrary, the three F 0 membrane subunits Atp6p, Atp17p (subunit f), and Atp4p exhibited dramatically reduced levels in the pellet fraction of the ⌬oxa1 strain (Fig. 2A). Organic solvent extraction of the F 0 subunits showed that Atp9p was decreased by ϳ40% in the ⌬oxa1 strain (Fig. 3B).

Altogether, these data clearly establish that there is a selective decrease in the accumulation of the membrane subunits of Cox and F 0 -ATPase in the absence of Oxa1p. It was previously shown that the mitochondrially encoded subunits are still synthesized in the absence of Oxa1p (4,11). Using a PhosphorIm- ager, the synthesis level of the Cox and ATPase mitochondrial subunits was quantified after 35 S labeling using the mitoribosomal protein Var1p as an internal control. The synthesis of Cox1p, Cox2p, and Atp6p was diminished by ϳ50%, whereas the synthesis of Cox3p and Atp9p was not affected in the absence of Oxa1p (data not shown). Thus, the fact that these subunits do not accumulate in the mitochondrial membranes or at a reduced level despite significant synthesis indicates that they are prone to degradation.

The Proteases Afg3p-Rca1p and Yme1p Are Not Responsible for the Degradation of Cox Subunits in the ⌬oxa1 Strain-To assess the possible roles of the two inner membrane AAA proteases Afg3p-Rca1p and Yme1p in the membrane subunit degradation occurring in the absence of Oxa1p, we inactivated the genes encoding these proteases in a ⌬oxa1 background. The single mutants ⌬afg3, ⌬rca1, and ⌬oxa1 are respiratory-deficient, whereas ⌬yme1 exhibits only a thermosensitive respiratory growth. The double mutants ⌬oxa1⌬afg3, ⌬oxa1⌬rca1, and ⌬oxa1⌬yme1 were all respiratory-deficient, showing that the lack of protease does not fully compensate for the absence of Oxa1p. The ⌬oxa1 mutant does not accumulate rho Ϫ mitochondrial mutants. In the ⌬afg3 and ⌬oxa1⌬afg3 strains, the percentage of rho Ϫ mutants was ϳ15% under standard conditions of culture.

As shown in Table I, cytochrome c oxidase activity was still defective in the double mutants, and the membrane subunits Cox1p, Cox2p, Cox3p, Cox5p, and Cox6ap were still degraded (Fig. 1B and data not shown). In addition, the extramembrane subunits, Cox4p and Cox6p, also displayed the same pattern as in the ⌬oxa1 single mutant (Fig. 1A and data not shown). Similarly, Cox2p remained undetectable in whole cell extracts from ⌬oxa1⌬rca1 and ⌬oxa1⌬rca1⌬afg3 strains (data not shown), suggesting that the accumulation of Cox1p, Cox3p, and Cox4p is also affected since the accumulation of these four subunits is interdependent (43). Thus, the absence of Afg3p-Rca1p or Yme1p does not seem to restore the stability of the Cox subunits.

Since it has been proposed that Afg3p-Rca1p could have a dual protease and chaperone activity (26,27), the subunit degradation occurring in the ⌬oxa1⌬afg3 double mutant could be due to the lack of the chaperone function of Afg3p-Rca1p. The absence of Afg3p-Rca1p leads to defects in respiratory complex assembly (17,18,25) that can be complemented by a proteolytically inactive variant of Afg3p in which glutamic acid 559 of the proteolytic site is changed to a glutamine (21,27). Thus, we have constructed a strain (RF0-7C) with the double inactivation ⌬oxa1⌬afg3 and a plasmid carrying the afg3-E559Q allele (21). In this strain, the chaperone activity of Afg3p should still be functional, whereas the protease activity of the Afg3-Rca1p complex is abolished. We found that Cox2p remained undetectable in whole cell extracts of RF0-7C, showing that the Cox2p degradation in the ⌬oxa1⌬afg3 strain is not due to the absence of the chaperone activity of Afg3p-Rca1p. In conclusion, Afg3p-Rca1p is not responsible for the degradation of membrane subunits of Cox that occurs in the absence of Oxa1p.

F 0 -ATPase Membrane Subunits Defective in the ⌬oxa1 Strain Are Stabilized in the Double Mutant ⌬oxa1⌬yme1-The ATPase activities of the double mutants ⌬oxa1⌬afg3 and ⌬oxa1⌬yme1 were compared with those of the corresponding wild-type and single mutant strains (Table I). Although the total ATPase activity was not significantly diminished in the various strains, the oligomycin-sensitive ATPase activity was strongly decreased in the ⌬oxa1, ⌬afg3, and ⌬oxa1⌬afg3 strains. Surprisingly, whereas Yme1p has not to date been reported to have a role in the degradation of ATPase subunits, we found that the inactivation of the YME1 gene in the ⌬oxa1 strain restored an oligomycin-sensitive ATPase activity, i.e. ⌬oxa1⌬yme1 reached 80% of the wild-type activity (Table I). Thus, the defect in the formation of the F 1 F 0 complex occurring in the ⌬oxa1 mutant was restored in the ⌬oxa1⌬yme1 strain since the oligomycin-sensitive ATPase activity reflects a well FIG. 2. Accumulation of ATPase subunits in mitochondrial membranes. Western blot analysis of mitochondrial proteins was performed as described in the legend to Fig. 1. S, supernatant; P, pellet. The blots were probed with polyclonal antibodies raised against the F 1 ␤-subunit (Atp2p), the oligomycin sensitivity-conferring protein subunit (Atp5p), and F 0 subunits (Atp6p, Atp17p, and Atp4p). Atp6p is a mitochondrially encoded subunit, whereas the others are nuclearly encoded. A, wild type (WT; CW04) and ⌬oxa1 (NBT1); B, ⌬yme1 (PHT7) and ⌬oxa1⌬yme1 (PHT8). FIG. 3. Organic solvent extraction of the F 1 -ATPase sector and of the F 0 subunit Atp9p. The F 1 sector and Atp9p were extracted by chloroform and chloroform/methanol, respectively (see "Experimental Procedures"). Proteins were fractionated on 15% SDS-polyacrylamide gels. A, F 1 subunits were transferred to nitrocellulose, and the blots were probed with antibodies against Atp1p, Atp3p, and Atp16p. B, the Atp9p subunit of F 0 was revealed by silver staining (48). The wild-type (WT; CW04) and ⌬oxa1 (NBT1) strains were used.

TABLE I Activities of Cox and ATPase complexes

Measurements of cytochrome c oxidase and ATPase activities were made on purified mitochondria. The values are an average of measurements obtained from two separate mitochondrial preparations. They are expressed as a percentage of the wild-type (WT) strain with an S.D. of ϳ10%. The cytochrome c oxidase activity of the wild-type strain is 770 nmol of oxidized cytochrome c/min/mg of protein. The specificity of cytochrome c oxidase activity was tested by addition of 4 M KCN. The total ATPase activity of the wild-type strain is 1.57 mol of P i /min/mg of protein, and the oligomycin-sensitive activity is 1.43 mol of P i / min/mg of protein. assembled F 1 F 0 complex. In addition, Western blot analysis showed that the accumulation of the F 0 membrane subunits Atp6p, Atp17p, and Atp4p, which was strongly diminished in the ⌬oxa1 mutant and not affected in the ⌬yme1 mutant, was restored in the double mutant ⌬oxa1⌬yme1 (Fig. 2B). On the contrary, the accumulation of these subunits was not restored in the ⌬oxa1⌬afg3 strain carrying the afg3-E559Q plasmid (RF0-7C) (data not shown). Thus, the absence of Yme1p restores the F 0 -ATPase subunit accumulation, which is defective in the absence of Oxa1p. Simultaneous Inactivation of AFG3 and YME1 Is Lethal-Since the Cox subunit stability was restored neither by YME1 nor by AFG3-RCA1 single inactivations, it was tempting to test the effect of the double inactivation. To construct the triple mutant ⌬oxa1⌬afg3⌬yme1, we crossed the ⌬oxa1⌬afg3 double mutant (F01-6C) with the ⌬yme1 strain (PHT7) and dissected the asci. However, no spore carrying the inactivation of both AFG3 and YME1 germinated. Similar results were obtained by crossing the ⌬afg3 strain (or ⌬rca1) with the ⌬yme1 strain, suggesting that the simultaneous absence of Afg3p-Rca1p and Yme1p is lethal whether OXA1 is deleted or not.

To test if this lethality was specifically due to the absence of the protease function of Afg3p-Rca1p, we crossed the ⌬afg3 strain, which expresses the proteolytically inactive variant afg3-E559Q (AFG3-2) on a URA3 plasmid, with the ⌬yme1 strain (PHT9). After dissection, all the URA3 ϩ spores carrying the plasmid were viable. We then forced the loss of the plasmid by growing the cells on uracil-containing medium supplemented with 5-fluoroorotic acid, which is toxic for uracil prototrophs; thus, cells that lose the plasmid and become uracil auxotrophs are able to grow on 5-fluoroorotic acid. The single mutant strains were able to grow on 5-fluoroorotic acid, but the double mutant ⌬afg3⌬yme1 (YA1-2C) was not (Fig. 4), showing that it cannot lose the plasmid. Since the plasmid expresses a proteolytically inactive variant of Afg3p, this shows that the protease activity is not the essential function.

DISCUSSION

In this study, we show that Oxa1p is specifically required for the stability of the membrane subunits of the Cox and ATPase complexes. Such specificity is consistent with the inner membrane location of Oxa1p and with a role in export and/or assembly of the membrane subunits of these two complexes. We then tested whether the unexported and/or unassembled membrane subunits could be the target of the known inner membrane protease complexes Yme1p and Afg3p-Rca1p.

Concerning the Cox subunits, Cox2p is a known substrate of Yme1p (20,23,24), and the complex Afg3p-Rca1p has been shown to participate in the degradation of Cox1p and Cox3p (21,27). Since pre-Cox2p export is blocked in the ⌬oxa1 mutant (11)(12)(13) and Yme1p is active in the intermembrane space, it is not surprising that Yme1p does not degrade pre-Cox2p in the ⌬oxa1 mutant. However, the fact that the inactivation of neither YME1 nor AFG3 could restore pre-Cox2p or the other Cox subunit stability suggests that either another protease is responsible for the degradation of these subunits or that they can be degraded by Yme1p and Afg3p-Rca1p. We rather favor the first hypothesis since the Cox subunits are still unstable when both Yme1p and Afg3p protease activities are blocked. It has been shown that the assembly of Cox1p, Cox2p, Cox3p, and Cox4p is interdependent (43). For example, in a cox2 mutant, the accumulation of the three other subunits is reduced. A specific pre-Cox2p export defect could then explain the general instability of most of the other Cox subunits found in ⌬oxa1 strains. However, the fact that the suppressor mutations that compensate for the absence of Oxa1p fully compensate for the maturation defect of Cox2p but only partially restore Cox activity (10) suggests that Oxa1p is not required only for Cox2p export. Whether the primary function of Oxa1p in Cox biogenesis is linked to export of membrane subunits and/or their assembly still remains an open question, both processes being tightly linked.

As far as the ATPase is concerned, we found that the F 0 membrane subunits are stabilized in the absence of Yme1p. Cross-linking experiments have shown that the subunits Atp4p, Atp6p, and Atp17p display accessible targets in the intermembrane space (41). However, it is difficult to determine whether they all represent true targets of Yme1p or whether only one subunit is degraded by this protease and exerts a protective effect upon the others. Nevertheless, the restoration of oligomycin-sensitive ATPase activity in the ⌬oxa1⌬yme1 double mutant shows that the increased stability of the ATPase membrane subunits allows significant translocation and assembly to occur. This result suggests that Oxa1p is not essential for the export of the ATPase subunits.

It has been proposed that the assembly of the three mitochondrially encoded subunits of the F 0 sector (Atp6p, Atp8p, and Atp9p) occurs first and is required for the subsequent assembly of Atp4p and then Atp5p (44 -46). We found that Atp6p and Atp4p levels are dramatically reduced in the ⌬oxa1 strain, whereas Atp9p and Atp5p levels are not. For Atp9p, our results can be related to the data showing that Atp9p is associated with the F 1 sector independently of the other F 0 subunits (44). For Atp5p, our data suggest that its accumulation is independent of the formation of the F 0 complex. In accordance with these results, we found that Atp5p was also stable in rho 0 cells (data not shown), which are devoid of mitochondrial DNA and therefore of mitochondrially encoded subunits of F 0 .

Finally, we have shown that the simultaneous absence of Afg3p (or Rca1p) and Yme1p is lethal and that viability is restored by the introduction of the proteolytically inactive variant of Afg3p. This suggests that Afg3p-Rca1p and Yme1p have an overlapping essential function that is probably due to the chaperone-like function. Alternatively, the cell viability might require both protease and chaperone activities to be functional. Although Oxa1p partially compensates for the AFG3 inactivation and could also display a "chaperone-like" function, the lethality of the double inactivation of AFG3 and YME1 cannot be cured by overexpressing the OXA1 gene (data not shown). It has been shown that the growth of ⌬yme1 rho Ϫ cells is severely affected (47). However, the lethality of the double mutant ⌬afg3⌬yme1 is probably not simply due to the accumulation of rho Ϫ mutants since the percentage of rho Ϫ mutants is ϳ15% in the ⌬afg3 mutant. In S. cerevisiae, respiratory function is dispensable, but the integrity of the mitochondrial compartment is essential. Thus, it is tempting to propose that Afg3p-Rca1p and Yme1p would be involved in the biogenesis of both respiratory complexes and other protein complexes controlling mitochondrial compartment integrity, whereas Oxa1p would play a role only in export and assembly of the membrane subunits of respiratory complexes.

FIG. 1 .

 1 FIG.1. Accumulation of Cox subunits in mitochondrial membranes. 300 g of mitochondrial proteins were treated with sodium carbonate; the supernatants (S; soluble fraction) and pellets (P; membrane fraction) were separated on 12 or 15% SDS-polyacrylamide gels and then transferred to nitrocellulose. Western blots were probed with monoclonal antibodies raised against various Cox subunits. Cox1p, Cox2p, and Cox3p are mitochondrially encoded subunits, whereas the others are nuclearly encoded subunits. A, wild type (WT; CW04) and ⌬oxa1 (NBT1); B, ⌬oxa1⌬afg3 (F01--6C) and ⌬oxa1⌬yme1 (PHT8) strains. See "Experimental Procedures" for complete genotypes.

FIG. 4 .

 4 FIG. 4. Simultaneous inactivation of AFG3 and YME1 is lethal.The two single mutants and the double mutant carrying the YepAfg3-2 plasmid were patched on uracil-containing media without (Ϫ5FOA) or with (ϩ5FOA) 1 mg/ml 5-fluoroorotic acid. Cells were incubated for 3 days at 28 °C. The ⌬yme1 (PHT9 ϩ YepAfg3-2), ⌬afg3 (AFG3-2), and ⌬afg3⌬yme1 (YA1-2C) strains were used.

Acknowledgments-We thank Drs. R. A. Capaldi (University of Oregon) and R. O. Poyton (University of Colorado) for the gift of antisera and Drs. T. D. Fox (Cornell University), L. A. Grivell (University of Amsterdam), and A. Tzagoloff (Columbia University) for the gift of strains or plasmids. We also thank Drs. N. Bonnefoy, O. Groudinsky, A. Sainsard-Chanet, C. J. Herbert, and Y. Saint-Georges for critical reading of the manuscript and N. Bonnefoy and C. J. Herbert for checking the English.

* This work was supported in part by a grant from the Association Franc ¸aise contre les Myopathies. Supported by a grant from the Ministe `re de l'Education Nationale, de l'Enseignement Supe ´rieur, de la Recherche, et de l'Insertion Professionnelle.