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Dual cell wall/mitochondria localization of the ‘SUN’ family proteins

Gisèle Velours, Catherine Boucheron, Stéphen Manon, Nadine Camougrand
DOI: http://dx.doi.org/10.1111/j.1574-6968.2002.tb11046.x 165-172 First published online: 1 February 2002

Abstract

The Saccharomyces cerevisiae SUN family gene products, namely Sim1p, Uth1p, Nca3p and Sun4p, show a high degree of homology among themselves and are closely related to β-glucosidase of Candida wickerhamii; however, these proteins do not bear such an activity. Dithiothreitol-treatment of intact cells induces the release of Uth1p, Sun4p and Sim1p from the cell wall. These highly glycosylated proteins are thus non-covalently bound to the cell wall. Two of them, Uth1p and Sun4p, have also been found in mitochondria. Sub-localization experiments show that Uth1p is inserted in the outer mitochondrial membrane and that Sun4p is preferentially a matrix protein. The physiological significance of this double localization is discussed in relation to the roles of these proteins in different cellular processes, namely mitochondrial biogenesis and cell septation.

Keywords
  • Protein localization
  • Cell wall
  • Mitochondria
  • Saccharomyces cerevisiae

1 Introduction

From different approaches, a new gene family termed the SUN family (SIM1, UTH1, NCA3 and SUN4) has been identified, the products of which are highly homologous. The remarkable feature of the four gene products is that they share a common C-terminal domain of 258 amino acids bearing 75–85% identity. These C-terminal regions comprise a putative Fe-binding domain consisting in Cys residues organized in a Cys-X5-Cys-X3-Cys-X24-Cys motif.

Despite this remarkable homology, the SUN family gene products are involved in various cellular functions. SIM1 gene (start independent mitosis) is involved in the regulation of CDK activity, as the null mutant Δsim1 allows for a second round of DNA replication in a strain lacking Clb1–Clb4 [1]. UTH1 is a yeast-aging gene that has been isolated on the basis of a better stress resistance and longer life span of mutants [2,3]. It was further shown to participate in both oxidative-stress response [4] and mitochondrial biogenesis [5]. NCA3 was cloned as a multicopy suppressor of a defect in mitochondrial synthesis of subunits 6 and 8 of the Fo sector of the ATP synthase, by acting at the maturation step of the co-transcript ATP8-ATP6 (formerly aap1-oli2) [6]. SUN4 was first identified by the yeast-sequencing effort [7]. More recently, Sun4p was found to be a dithiothreitol (DTT)-extractable component of the cell wall [8] and we further evidenced its involvement in cell septation [9].

Cell wall is the external envelope that surrounds yeast cells outside the plasma membrane. This structure is essential for maintaining cell morphology and protecting cells from aggressions from the external environment. Its composition changes in response to different environmental conditions [10]. The major components of the cell wall are β-glucans (resulting from β-1,3 and/or β-1,6 linkages), mannoproteins and chitin. On the basis of the methods used for their extraction, cell wall mannoproteins can be divided in three groups: (1) mannoproteins extractable by sodium dodecyl sulfate (SDS) or by reducing agents, (2) glucanase extractable mannoproteins and (3) proteins that remain in cell wall after SDS-extraction but that can be extracted by mild alkali treatment.

All the SUN family gene products are closely related to a β-glucosidase of Candida wickerhamii[11]. In addition, two proteins of the fission yeast Schizosaccharomyces pombe, namely psu1p [12] and a hypothetical protein identified in the cosmid c2G2, were recently found to also possess a C-terminal domain closely related to that of SUN family.

Here we investigate the localization of three proteins of this family, Uth1p, Sun4p and Sim1p. Like Sun4p, Uth1p and Sim1p were found to be cell wall proteins extractable by DTT-treatment of intact cells. In addition, Uth1p and Sun4p were found to have a mitochondrial localization. These and previous observations, suggest the existence of a regulatory link between mitochondria and cell wall functions.

2 Materials and methods

2.1 Strains and media

The yeast strains used in this study are listed in Table 1. Cells were grown aerobically at 28°C in the following medium: 0.175% Yeast Nitrogen Base without amino acids or ammonium (Difco), 0.5% ammonium sulfate, 0.1% potassium phosphate, 0.2% Drop mix, 0.01% leucine, and tryptophan, histidine, uracil, lysine or adenine, depending on auxotrophic requirements. Carbon sources were 2% glucose (YNBglu), 2% galactose+0.5% raffinose (YNBgal/raf) or 2%dl-lactate (YNBlac).

View this table:
Table 1

List of strains used in this study

W303-1B/AMATa, ade2, his3, leu2, trp1, ura3, can1
Δuth1 pgal UTH1MATa, ade2, his3, leu2, trp1, ura3, can1, uth1::TRP1+plasmid
pYES2/GS/UTH1
Δsun4 pgal SUN4MATa, ade2, his3, leu2, trp1, ura3, can1, sun4::kan-MX4+plasmid
pYES2/GS/SUN4
Δsun4 pgalΔ1-24sun4MATa, ade2, his3, leu2, trp1, ura3, can1, sun4::kan-MX4+plasmid
pYES2/GS/Δ1-24sun4
Δsim1 pgal SIM1MATa, ade2, his3, leu2, trp1, ura3, can1, sim1::LEU2+plasmid
pYES2/GS/SIM1
Δuth1 pvUTH1MATa, ade2, his3, leu2, trp1, ura3, can1, uth1::TRP1+plasmid
pGRL1/pvUTH1

2.2 Construction of UTH1, SUN4 and SIM1 fusions to the V5 epitope

Escherichia coli strain DH5α was used for the construction and the amplification of plasmids. The pYES2/GS/SUN4 vector was purchased from Invitrogen (The Netherlands). The SUN4 gene, fused to the V5 epitope and the His6 tag at its C-terminal end, is placed under the control of the GAL1/GAL10 promoter.

pYES2/GS/SUN4 was used to construct plasmids expressing UTH1, SIM1 and a Δ1-24 mutant of SUN4 (see below), tagged at their C-terminal end with the V5 epitope and the His6 tag. The corresponding genes were amplified by PCR using primers containing a 5′-EcoRI site and a 3′-BstEII site (primers: 5′-CGGAATTCATGTGTTTCCTTCTCGAGA-3′ and 5′-AGGGTGACCGTAGAAGACAAAGTTAGCA-3′ for UTH1; 5′-CGGAATTCATGAAATTCTCAACTGCC-3′; and 5′-GAGGGTGACCATTGTATAAGACGAAATG-3′ for SIM1 and 5′-CGGAATTCATGTATGCGGCTGATATTGACACAGG-3′ and 5′-GAGGGTGACCGTTGTATAGAACAAACTTAGC-3′ for Δ1-24 SUN4) (EcoRI and BstEII sites are indicated in bold characters). The PCR products were subsequently cloned into the pYES2/GS/SUN4 vector digested by the same restriction enzymes to give the following plasmids: pYES2/GS/UTH1, pYES2/GS/SIM1 and pYES2/GS/Δ1-24 sun4. All constructions were verified by sequencing.

2.3 Localization of Uth1p using GFP fusion

The UTH1 gene and its 5′-flanking region containing its promoter was amplified by PCR using the following primers: 5′-GCGGGATCCTGATTGATGACAAGCGTAGAA-3′ and 5′-CGCGGATCCGTAGAAGACAAAGTTAGCAGA-3′ containing a BamHI site (bold characters). The PCR product was digested by BamHI and was subsequently cloned into the corresponding site of the pGRL1 vector (gift of Dr B. Daignan-Fornier, Bordeaux). This construct (pGRL1/pVUTH1), verified by sequencing, encodes a chimeric protein with the green fluorescent protein (GFP) fused at the C-terminus of Uth1p expressed under its own promoter. Localization of the fusion protein was observed in living cells of strains Δuth1 transformed with pGRL1/pVUTH1 or pGRL1 as a control and grown on different carbon sources. The cells were viewed with a Leica DMRXA microscope using a 100× immersion objective and (i) an excitation wavelength of 450–490 nm with a barrier filter of 515–560 nm for GFP; (ii) an excitation wavelength of 530–595 nm with a barrier filter superior to 615 nm for rhodamine 123.

2.4 Molecular biology techniques

Yeast transformations were done by the lithium acetate method [13]. Transformants were selected on YNBglu medium supplemented with the appropriate auxotrophic requirements.

2.5 Proteins extraction

Cells were grown in YNBglu medium, and then transferred to a YNBgal/raf medium during 12 h to induce V5-tagged proteins expression. Cells were harvested in mid-exponential growth phase. 2×107 cells were washed, resuspended in 0.5 ml water, and added with 0.05 ml of a 1.85 M NaOH, 3.5%β-mercaptoethanol mixture. After a 10-min incubation on ice, proteins were precipitated by adding 0.05 ml of 3 M trichloroacetic acid (TCA) and a further 10 10-min incubation on ice. After centrifugation, the pellet was solubilized in a 1/1 (v/v) mixture of 10% SDS and sample buffer (0.1 M Tris, 2% SDS, 2%β-mercaptoethanol, 25% glycerol, 0.002% bromophenol blue, pH 7.0). DTT extraction was done on 15×107 cells washed twice with water and then incubated with 2 mM DTT for 2 h at 4°C. After centrifugation, the supernatant was concentrated and analyzed by SDS–polyacrylamide gel electrophoresis (PAGE).

2.6 Mitochondria purification

Mitochondria were isolated and further purified on a sucrose density gradient as described in [14]. For hypoosmotic treatment, 2 mg mitochondria was suspended in 1 ml of a 20 mM Na–HEPES buffer, pH 7.4, added with 0.5% SAB and 100 μg ml−1 proteinase K, and incubated for 15 min at 0°C. The reaction was stopped by adding 1 mM phenylmethylsulfonyl fluoride. Proteins were precipitated with 0.3 M TCA, pelleted, resuspended in sample buffer and separated by SDS–PAGE. Mitochondrial outer membranes (OMs) and mitochondrial inner membranes (IMs) were isolated as described in [15]. Extraction of mitochondria with sodium carbonate was performed as described in [15].

2.7 Zymolyase treatment

Cells were harvested in mid-exponential growth phase. 5×107 cells were washed, resuspended in 25 μl digestion buffer (1.35 M sorbitol, 10 mM citric acid, 30 mM Na2HPO4, 1 mM EGTA pH 5.8), and added with 25 μg zymolyase 20T. After a 10-min incubation at 28°C, cells were washed and proteins were extracted as described above.

2.8 Purification of Sun4p by Ni-NTA affinity chromatography

Δsun4 pgalSUN4 strain was grown on a YNBgal/raf medium and harvested in mid-exponential growth phase. Mitochondria were isolated and purified on a density sucrose gradient. They were resuspended (10 mg ml−1) in a 50-mM sodium phosphate buffer (pH 8.0) added with 0.3 M NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 0.75% Triton X-100, anti-proteases cocktail (Complete EDTA-free, Boehringer) and solubilized for 20 min at 4°C. After centrifugation (100 000×g for 35 min), the supernatant was dialyzed for 18 h against the same buffer without Triton X-100. The dialysate, containing the His6-tagged Sun4p, was incubated with Ni-NTA resin (Quiagen) in a batch procedure. The batch was equilibrated with a 50-mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole. Proteins bound to Ni-NTA were eluted with a step gradient of imidazole. The collected fractions were tested for the presence of Sun4p. Under these experimental conditions, Sun4p was essentially eluted with 75 mM imidazole. This fraction was loaded on a POROS HQ (Applied Biosystems) high capacity strong anion exchange column (FPLC) equilibrated with a 20-mM Tris–HCl buffer (pH 8.5) containing 20 mM NaCl and eluted with a 50–300-mM linear NaCl gradient in 20 mM Tris–HCl buffer (pH 8.5). Different proteic fractions were collected and analyzed for Sun4p by SDS–PAGE and Western blotting.

2.9 Electrophoresis and blotting

SDS–PAGE was performed by the method of Laemmli [16]. After electrotransfer onto ProBlott membranes (Applied Biosystems), blots were probed with antibodies against V5 epitope (Invitrogen, dilution 1:5000) or Uth1p (Eurogentec, dilution 1:1000). Then, membranes were incubated with horseradish peroxidase-labelled antibodies (Jackson Immunoresearch, 1:5000) and revealed with an enhanced chemoluminescence kit (Amersham Pharmacia Biotech.). Automated sequence analyses were done on a gas-phase automatic sequencer (Applied Biosystems 470A) and phenylthiohydantoin-amino acids were identified with an on-line high-performance liquid chromatography system. Sequence cycles were run according to the standard protocol provided by the manufacturer.

3 Results

3.1 Cell wall localization

Sun4p exhibits a strong homology to cell wall-glucanases and was co-isolated with six other proteins as cell wall-components, easily released from intact cells by DTT-treatment [8]. This cell wall localization can be related to the defects observed in sun4Δ cells: for example, in contrast to wild-type, bud scars did not appear in the same depth of focus as the cell wall and this is thought to account for a modification in the cell wall mechanical strength [9]. More precisely, the following observations were reported: (a) sun4Δ cells were delayed in cell separation; (b) although they exhibit the same α-factor sensitivity as the wild-type, mat a, sun4Δ mutant was shown to develop shmoos (apperition of a specific deformation of yeast cells after an alpha-factor treatment) on unseparated G1 cells; (c) sun4Δ cells exhibited a partial resistance to lysis by β-1,3-glucanases. The UTH1/SUN4 double inactivation further increased the delay in cell separation observed in sun4Δ[9].

Like Sun4p, both Uth1p and Sim1p were released from intact cells by DTT-treatment (Fig. 1A, lanes 2). Such treatment of intact cells with reducing agents releases non-covalently linked cell wall proteins as well as proteins that are anchored to this structure via disulfide bridges. The proteins migrated on SDS-PAGE as diffuse bands of much greater apparent molecular mass than those predicted by their amino-acids sequences. These pronounced differences in size of the calculated proteins (43 414 kDa, 46 916 kDa and 48 046 kDa for Sun4p, Uth1p and Sim1p respectively) compared to the actual measured one (96 000 kDa for Sun4p, 60 000 kDa for Uth1p, 120 000 kDa and a band higher than 160 000 kDa for Sim1p), suggest a high degree of glycosylation. These proteins actually possess potential N-glycosylation sites at position 404 for Sun4p, 79 and 83 for Uth1p and 422 for Sim1p. To confirm the glycosylation state of these proteins, cells were grown in the presence of tunicamycin or treated with zymolyase and proteins extractions were performed. After zymolyase treatment (Fig. 1B, lane 2) or tunicamycin treatment (not shown) bands having the theoretic size of unmodified proteins were evidenced.

Figure 1

Cell wall localization of proteins of the SUN family. A: Cells were grown on YNBgal/raf medium. Total proteins were extracted from the equivalent of 107 cells. 15×107 cells were subjected to 2 mM DTT treatment for 2 h at 4°C. Then, total extract and DTT supernatant were analyzed by SDS–PAGE. After blotting, proteins were visualized with V5 antibodies. Lanes 1: total extract; lanes 2: DTT extracts. B: Zymolyase treatment. a: Δsun4 pgalSUN4 strain; b: Δsim1 pgal SIM1 strain; c: Δuth1 pgalUTH1strain. Cells were grown on YNBgal/raf medium and the equivalent of 107 cells were treated or not with 10 mg g−1 dry weight zymolyase 20T for 10 min and then total proteins were extracted and analyzed by SDS–PAGE. After blotting, proteins were revealed with V5 antibodies. Lanes 1: no treatment; lanes 2: zymolyase treatment.

All SUN family proteins are closely related to a β-glucosidase of C. wickerhamii based on protein sequence homology. Therefore, we investigated whether these proteins actually exhibited such an activity. We measured β-glucosidase activity in different strains: wild-type, strains inactivated for UTH1, SIM1 and SUN4 and strains inactivated both for UTH1 and SUN4, for UTH1 and SIM1 and for SUN4 and SIM1 genes. No significant difference was observed between all these strains and the activity measured in the doubly inactivated strains was actually slightly increased (not shown). Thus no β-glucosidase activity could be associated to these proteins.

Disruption of the SUN family genes did not affect the viability and morphology of the cells, nor their mating efficiency, nor their sensitivity to agents that interfere with the synthesis of the cell wall (calcofluor white) or degrade it (congo red); disruption of the SUN4 gene only delayed the last step of the cell cycle that is the separation between mother and daughter cells [9].

The lack of any conventional N-terminal signal sequence in these proteins suggests the involvement of a non classical export machinery in their targeting to the cell surface, as described for several other fungal and higher eukaryotic proteins [17]. Other proteins lacking a signal peptide such as some heat shock proteins belonging to the Hsp70 family have also been localized in the cell wall of both Saccharomyces cerevisiae and Candida albicans[18]. Sun4p was purified from DTT extracts by using the His6-tag and purification on Ni-NTA resin. The N-terminus part of the purified protein was sequenced. The following sequence was obtained: YAADIDT. These results show that the 24 first amino acids are cleaved during the targeting of the protein to the cell surface. We constructed a strain (Δ1-24 Sun4) that expresses Sun4p deprived of these 24 amino acids to evaluate if this sequence is required to target the protein to the cell wall. Truncated Sun4p is expressed (Fig. 2b, lane 1) but only two bands can be visualized (96 and 60 kDa), each representing about 50%, suggesting different states of glycosylation. Like the native protein, the truncated protein of higher mass (96 kDa) was found in the supernatant after DTT-treatment of intact cells (Fig. 2b, lane 2), suggesting that the cell wall localization of the mature glycosylated protein is not modified. We conclude that this sequence is not necessary to target Sun4p to the cell wall but its absence perturbs the glycosylation step.

Figure 2

Localization of the Δ1-24Sun4p. The Δsun4 pgalΔsun4 (a) and the Δsun4 pgalΔ1-24sun4 (b) strains were grown on a YNBgal/raf medium. Lanes 1: Whole cell extracts were prepared as in Section 2. Lanes 2: DTT extracts from intact cells were obtained as described in Fig. 1A.

3.2 Mitochondrial localization of Sun4p

From databases, Sun4p is predicted to contain a mitochondrial transit peptide, unlike the other members of the SUN family. We therefore investigated whether this protein is also localized in the mitochondrial fraction. SDS–PAGE/Western blot analyses of a whole cell extract from Δsun4galSUN4 strain, shows that the protein exists under two forms: a major one corresponding to the 96 kDa glycosylated form and a minor band migrating at 38kDa, closer to the theoretic size of the non-modified polypeptide chain (Fig. 3A). Mitochondria from this strain were isolated and purified on a density sucrose gradient. The presence of Sun4p was detected in this highly pure mitochondrial fraction (Fig. 3B, lane 1). After carbonate treatment, this protein was preferentially found in the supernatant fraction showing that it is not integrated in membranes (Fig. 3D). Purification of this mitochondrial form of Sun4p was performed by chromatography on Ni-NTA resin and elution with a gradient step of imidazole. The majority of Sun4p was found in the 50- and 75-mM imidazole fractions (Fig. 3B, lanes 2–5). The last step of purification was a chromatography on a POROS HQ column (not shown). The peak eluted at 53 mM NaCl corresponded to pure Sun4p, as shown by Western blot (Fig. 3B, lane 6) and amidoblack staining (not shown). This stained band was cut out and mass spectrometry analysis confirmed the identity of Sun4p.

Figure 3

Mitochondrial localization of Sun4p. A: Whole cell extract from Δsun4 pgalSUN4 strain (equivalent to 2×107 cells). B: Lane 1: purified mitochondria; lanes 2–5: different fractions eluted by the gradient step of imidazole (2: 20 mM; 3: 50 mM; 4: 75 mM; 5: 150 mM). C,D: The Δsun4 pgalSUN4 and the Δsun4 pgalΔ1-24sun4 strain were grown on a YNBgal/raf medium. Mitochondria were isolated and treated with NaCO3 0.1 M. Mitochondria (M), pellet (P) and supernatant (S) were analyzed on SDS–PAGE. Blots were probed with V5 and anti-Atp4 antibodies.

The same experiments were performed with the Δ1-24 Sun4 strain that expresses Sun4p deprived of the first 24 amino acids. Truncated Sun4p was not found in mitochondria (Fig. 3C). We conclude that the 24 first amino acids are necessary to target Sun4p to the mitochondria. Preliminary mass spectrometry experiments suggest that the mitochondrial form of Sun4p was glycosylated. We hypothesize that the transient passage of Sun4p in mitochondria could be necessary to cleave the signal sequence and to trigger the glycosylation process.

3.3 Mitochondrial localization of Uth1p

In order to study the cellular localization of Uth1p, we expressed a gene fusion by inserting GFP immediately upstream of the stop codon of the UTH1 gene. This fusion protein was expressed under the control of UTH1's own promoter. Fluorescence microscopy analysis of living cells allowed to detect the fusion protein as punctuate patches inside the cells. These patches co-localized with mitochondrial marker rhodamine 123 (Fig. 4A). The number of patches increased when the cells were grown on a carbon source such as lactate that requires oxidative metabolism and well-differentiated mitochondria. This behavior indicates a mitochondrial localization for Uth1p. This was further verified by investigating Uth1p localization after purification and fractionation of mitochondria. The Δuth1 pgalUTH1 strain was grown on YNBgal/lac medium and harvested in mid-exponential growth phase. Mitochondria were isolated and purified on a sucrose density gradient. Isolated mitochondria were highly pure, devoid of the ER markers Sec61 and Sss1 as well as the vacuolar marker ALP [13]. Uth1p was detected in this fraction (Fig. 4C, lane M). Then, mitochondria were subfractioned in mitochondrial OM- and mitochondrial IM-enriched fractions, that were analyzed for the presence of Uth1p (Fig. 4C). Uth1p was found in the OM fraction. As a control, ATP synthase subunit Atp4p was found in the IM fraction whereas porin (VDAC), an abundant protein present at the contact sites between the two membranes, was present in both fractions. When mitochondria were incubated in a hypotonic buffer in order to rupture the OM and then treated with proteinase K, porin and Uth1p were degraded whereas Cox2p was not (Fig. 4B). These results confirmed the presence of Uth1p in the mitochondrial OM.

Figure 4

Localization of Uth1p. A: Living cells (Δuth1 pvUTH1 strain) were grown on different carbon sources and were harvested in mid-exponential phase. Cells were incubated with 1 μM rhodamine 123 for 30 min and then visualized by microscopic fluorescence. Bars represent 1 μM. B,C: Localization in vitro of Uth1p. Mitochondria from Δuth1galUTH1 were isolated and purified on a density sucrose gradient. B: Mitochondria (2 mg ml−1) were suspended in a hypoosmotic buffer and treated (+PK) or not (–PK) with 100 μg ml−1 of proteinase K. C: Mitochondrial IMs and OMs were purified according to [14]. 150 μg proteins were separated on SDS–PAGE and analyzed for Uth1p, VDAC, Cox2p and Atp4p by using specific antibodies.

4 Discussion

The major cell wall components of S. cerevisiae are mannoproteins, β-glucans and chitin that provide cell protection against external stresses. Mannoproteins include reducing agent- extractable mannoproteins, glucanase-extractable mannoproteins and SDS-extractable proteins. Sun4p was found as a cell wall component [8]. In this paper we confirmed the cell wall-localization of this protein by DTT treatment, and further showed that two other members of the SUN family, Sim1p and Uth1p, exhibit a similar localization. In addition, we found that two of these proteins, Uth1p and Sun4p, have an additional mitochondrial localization.

Previous reports showed that Uth1p participates in the regulation of mitochondrial biogenesis [5]. Inactivation of UTH1 gene resulted in two effects: (i) a global decrease of mitochondrial proteins as compared to total cellular proteins; (ii) among respiratory enzymes, cytochrome aa3 content was more affected than cytochrome b content, suggesting a preferential decrease of cytochrome c oxidase as compared to bc1 complex. Since the mitochondria-specific phospholipid cardiolipin, mitochondrial DNA content and the average number of mitochondria on EM pictures were identical in wild-type and Δuth1 strains, it was hypothesized that the protein content of Δuth1 mitochondria was decreased but that the number of mitochondria remained closed to that of wild-type. Consequently Uth1p could play a role in the regulation of mitochondrial biogenesis, which is in accordance with its mitochondrial localization.

SUN family members are highly glycosylated when localized in the cell wall. They have significant homologies to glucanases, although they do not bear such activity. Among cell wall mannoproteins, most have an N-terminal secretion signal that ensures the export of proteins to the cell wall by the secretory pathway. Proteins lacking N-terminal secretion signal reach the cell wall via non-classical pathways. For example, factor a lacks a signal sequence and is secreted [19]. Furthermore, some cytosolic proteins such as enolase, phosphoglycerate kinase and members of the Hsp70 family have been found in S. cerevisiae and C. albicans cell walls [18,2025]. Reciprocally, proteins first identified as cell wall components, were recently shown to have also an intracellular localization. For example, Pir1p was initially isolated as a cell wall component [26,27]. This protein interacts with the C-terminal end of nuclease Apn1p, in the cytosol or the nucleus, and was shown to facilitate its transport into mitochondria to repair damaged DNA [28]. These results show that Pir1p has a double cell wall/intracellular localization. Pir1p bears no obvious organelle-targeting signal sequence. Gas1p provides another example of double cell wall/intracellular localization. Separation of yeast mitochondrial complexes by colorless native PAGE led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity. Gas1p, a cell wall protein [29] attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol anchor [30] was also found in this mitochondrial supramolecular complex [31]. This provides another indication that a relatively large number of proteins have a double mitochondria/cell wall localization.

The unexpected association of some known cellular genes with cell wall phenotypes emphasizes the value of genome-wide screens to define function and to examine global aspects of regulation in the yeast cell [32]. Proteins encoded by these genes can bear several functions, ranging from involvement in metabolism (for example glycolysis enzymes), mitochondrial function (for example Uth1p), transcription, translation and DNA repair (for example Pir1p). Another possible example of a global regulatory response is illustrated by mitochondrial defects that appear to alter yeast cell surface. IFM1, SMP2 and COX11 are all nuclear petite genes [3335] with cell surface phenotypes. Earlier literature on this theme has been overlooked [36,37]. The double cell wall/mitochondria localization of two members of the SUN family Uth1p and Sun4p, with phenotypes associated to cytokinesis for Sun4p and to mitochondrial biogenesis for Uth1p further highlights the existence of some regulatory link between mitochondrial function and the cell surface.

Acknowledgements

Authors thank Professor J.M. Schmitter for mass spectrometry experiments and Dr. S. Chaignepain for performing N-terminal sequences. This work was supported by grants from the Victor Segalen University of Bordeaux II, The Centre National de la Recherche Scientifique, the Conseil Régional d'Aquitaine and the Association pour la Recherche contre le Cancer.

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View Abstract