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Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome

2002; Springer Nature; Volume: 21; Issue: 1 Linguagem: Inglês

10.1093/emboj/21.1.43

1460-2075

Antoni Barrientos,

ATP Synthase and ATPases Research

Article15 January 2002free access Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome Antoni Barrientos Antoni Barrientos Department of Biological Sciences, Columbia University, New York, NY, 10027 USA Search for more papers by this author Daniel Korr Daniel Korr Department of Biological Sciences, Columbia University, New York, NY, 10027 USA Search for more papers by this author Alexander Tzagoloff Corresponding Author Alexander Tzagoloff Department of Biological Sciences, Columbia University, New York, NY, 10027 USA Search for more papers by this author Antoni Barrientos Antoni Barrientos Department of Biological Sciences, Columbia University, New York, NY, 10027 USA Search for more papers by this author Daniel Korr Daniel Korr Department of Biological Sciences, Columbia University, New York, NY, 10027 USA Search for more papers by this author Alexander Tzagoloff Corresponding Author Alexander Tzagoloff Department of Biological Sciences, Columbia University, New York, NY, 10027 USA Search for more papers by this author Author Information Antoni Barrientos1, Daniel Korr1 and Alexander Tzagoloff 1 1Department of Biological Sciences, Columbia University, New York, NY, 10027 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:43-52https://doi.org/10.1093/emboj/21.1.43 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info SHY1 codes for a mitochondrial protein required for full expression of cytochrome oxidase (COX) in Saccharomyces cerevisiae. Mutations in the homologous human gene (SURF1) have been reported to cause Leigh's syndrome, a neurological disease associated with COX deficiency. The function of Shy1p/Surf1p is poorly understood. Here we have characterized revertants of shy1 null mutants carrying extragenic nuclear suppressor mutations. The steady-state levels of COX in the revertants is increased by a factor of 4–5, accounting for their ability to respire and grow on non-fermentable carbon sources at nearly wild-type rates. The suppressor mutations are in MSS51, a gene previously implicated in processing and translation of the COX1 transcript for subunit 1 (Cox1) of COX. The function of Shy1p and the mechanism of suppression of shy1 mutants were examined by comparing the rates of synthesis and turnover of the mitochondrial translation products in wild-type, mutant and revertant cells. We propose that Shy1p promotes the formation of an assembly intermediate in which Cox1 is one of the partners. Introduction SHY1, the yeast homologue of the human SURF1 gene, codes for a mitochondrial protein necessary for full expression of respiration and cytochrome c oxidase (COX) (Mashkevich et al., 1997). Mutations in SURF1 are responsible for most cases of Leigh‘s syndrome (LS) associated with cytochrome oxidase deficiency (Tiranti et al., 1998; Zhu et al., 1998), a heterogeneous group of mitochondrial diseases with preferential neuropathological symptoms and morphological and histochemical defects in mitochondria (Leigh, 1951). Patients with Leigh’s syndrome have severely lowered levels of COX, although there are other variable biochemical defects associated with the disease (DiMauro and De Vivo, 1996; Rahman et al., 1996; Tiranti et al., 1998; Zhu et al., 1998). The similarity in the biochemical phenotypes of mitochondria from shy1 mutants and from Leigh patients implies a common function of the two gene products. The precise function of Shy1p and Surf1p is not known. Studies on Surf1p have focused on its presumed role in COX assembly. Mitochondrial COX, the terminal enzyme of the respiratory chain, has three redox centers that catalyze a sequential transfer of electrons from cytochrome c to molecular oxygen. COX is located in the mitochondrial inner membrane and in yeast is made up of 12 different subunits. The catalytic core of the enzyme consists of three subunits derived from mitochondrial genes. The other nine subunits are products of nuclear genes. Assembly of COX is a complicated process that requires the assistance of numerous nuclear gene products. An understanding of their functions is only now beginning to emerge, mainly as the result of studies of yeast mutants (Glerum et al., 1996; Hell et al., 2000, 2001; Souza et al., 2000). Unlike other assembly-defective strains of yeast that display a complete absence of COX (Glerum and Tzagoloff, 1998), shy1 mutants produce 10–15% fully assembled and functional COX. In addition to their lower COX content, shy1 mutants exhibit other alterations. For example, they have elevated levels of cytochrome c and their NADH cytochrome c reductase activity is two times higher than in wild type (Mashkevich et al., 1997). LS patients also have 10–30% of normal COX. Analysis of surf1 mutant cell lines by blue native gel electrophoresis indicates a partial block of COX assembly at an early step of the pathway, most likely before the incorporation of subunit II into the nascent intermediates composed of subunit I alone or subunit I plus subunit IV (Coenen et al., 1999; Tiranti et al., 1999). To understand better the role of Shy1p/Surf1p in COX assembly we have isolated revertants of shy1 mutants with nearly normal growth properties on respiratory substrates. The revertants have been analyzed genetically and shown to have extragenic nuclear suppressors. The respiratory activities of mutants and revertants indicate that rescue of the respiratory defect correlates with an increase in the mitochondrial concentration of COX. The suppressor was cloned from a plasmid library constructed from the nuclear DNA of the revertant and identified to be MSS51, a yeast gene previously implicated to play a role in translation of COX subunit 1 (Cox1) (Decoster et al., 1990). In vivo or in organello labeling of mitochondrial translation products revealed substantially reduced expression of Cox1 in the mutant. The cytochrome oxidase deficiency is partially restored by the suppressor, which is proposed to increase translation of Cox1, thereby compensating for a Shy1p-dependent step in assembly of the enzyme. Results Properties of shy1 mutants and revertants Both the point mutant W125 and the shy1 null mutant W303ΔSHY1/U2 (ΔSHY1) spontaneously convert to respiratory competence at a high frequency. When 107 cells are spread on rich glycerol medium (YEPG), revertant colonies appear after 4–5 days of incubation at 30°C. With prolonged incubation, the plates become overgrown with revertants (Figure 1A). Revertants also appear at 37°C, but they are fewer in number. Growth of two independent revertants (ΔSHY1/R1 and ΔSHY1/R2) on solid or in liquid YEPG is only slightly slower than the parental wild type (Figure 1B and C). The doubling time on glycerol was 2.4 h for the wild type, 3.3 h for the revertant and 28 h for the mutant. Figure 1.Growth properties of shy1 mutants and revertants. (A) The shy1 null mutant ΔSHY1 was plated at a density of 107 cells/plate on rich ethanol/glycerol (YEPG). The plates were photographed after 6 and 8 days of incubation at 30 and 37°C. (B) The respiratory-competent strain W303-1A, the shy1 null mutant ΔSHY1, and two independent revertants ΔSHY/R1 and ΔSHY/R2 were inoculated into liquid YEPG media and incubated with vigorous shaking at 30°C. Growth was monitored by absorbance at 600 nm. The doubling times for the different strains are indicated. (C) Serial dilutions of the respiratory-competent strain W303-1A, the shy1 mutant ΔSHY1, and three revertants ΔSHY/R1, ΔSHY/R2 and ΔSHY/R3 with the mss51 mutations indicated were spotted on YPD and YEPG plates and incubated at 30 and 37°C for 2–3 days. Download figure Download PowerPoint Crosses of two independent revertants, ΔSHY1/R1 and ΔSHY1/R2, to the shy1 null mutant produced respiratory-competent diploid cells, indicating that the mutations behave as dominant suppressors. The suppressors were ascertained to be nuclear by two criteria. Revertants were converted to ρo/ρ− derivatives by treatment with ethidium bromide. Panels of the ρo/ρ− clones were crossed to the null mutants. The diploid cells issued from these crosses had the same phenotype as the haploid revertants, attesting to the nuclear nature of the suppressors. This was confirmed by tetrad analysis of diploid cells obtained from crosses of the revertants to the ΔSHY1 mutant. The meiotic spore progeny from 16 complete tetrads all had the URA3 marker for the shy1 null allele. Two spores in each tetrad were respiratory competent, consistent with the presence of a dominant nuclear suppressor. Dissections of tetrads obtained from crosses of the revertants to the parental wild-type strains (Ura−) failed to show co-segregation of the respiratory competence, and uracil prototrophy indicated that neither suppressor is linked to SHY1. The presence of the suppressors in a wild-type nuclear background (20% of the progeny) had no effect on respiration (data not shown). Respiration in whole cells and in isolated mitochondria is partially restored in shy1 revertants The ΔSHY1 strain respires at 20% of the wild-type rate in whole-cell assays using galactose or glucose as substrates (Figure 2A). This respiration is inhibited 59% by antimycin A (AA) and 93% by KCN. The respiratory activity of the revertant is increased to 78% of wild type and is completely sensitive to AA and KCN. These high in vivo respiration rates explain the ability of the revertant to grow on non-fermentable carbon sources. Even though mutant cells have 20% residual respiration, this activity is only partially sensitive to AA. The reduced oxidative capacity combined with partial sensitivity to AA helps to explain the mutant's failure to grow on respiratory substrates. Identical results were obtained with the shy1 mutant containing partially deleted SHY1 (Mashkevich et al., 1997). Figure 2.Functional characterization of the shy1 null mutant and revertant. (A) In the left panel, mitochondria prepared from the wild type (W303-1A), from the shy1 mutant (ΔSHY1) and from the revertant (ΔSHY1/R1) were assayed polarographically for NADH oxidase (NADH oxid.). Respiration was also assayed in whole cells in the presence of glucose (Cell Resp.). The specific activities reported were corrected for AA-insensitive respiration. The bars indicate the mean ± SD from three independent sets of measurements. In the right panel, COX was assayed in frozen–thawed mitochondria (COX) and in mitochondria permeabilized with potassium deoxycholate (COX/DOC) by measuring oxidation of ferrocytochrome c at 550 nm. COX activity was also assayed polarographically by measuring the oxygen consumption rate in the presence of ascorbate plus TMPD. (B) Cytochrome spectra. Mitochondria were extracted at a protein concentration of 5 mg/ml with potassium deoxycholate under conditions that quantitatively solubilize all the cytochromes (Tzagoloff et al., 1975). Difference spectra of the reduced (sodium dithionite) versus oxidized (potassium ferricyanide) extracts were recorded at room temperature. The α absorption bands corresponding to cytochromes a and a3 have maxima at 603 nm (a). The maxima for cytochrome b (b) and for cytochrome c and c1 (c) are 560 and 550 nm, respectively. (C) Steady-state concentrations of COX subunits. Total mitochondrial proteins (30 μg) separated by 16.5% SDS–PAGE were transferred to nitrocellulose and probed with subunit-specific antibodies to COX subunits. Download figure Download PowerPoint Respiration was also assayed polarographically in isolated mitochondria by measuring the rates of oxygen consumption with NADH as substrate. AA-sensitive oxidation of NADH in ΔSHY1 mitochondria was 14% of wild type (Figure 2A). This rate was four times higher in the ΔSHY1/R1 revertant. Even so, overall oxidation of NADH in the revertant was still only 60% of the wild-type rate (Figure 2A). The possibility that the growth defect of shy1 mutants is due to a lesion in oxidative phosphorylation was excluded by measurements of the phosphorylation efficiency in isolated mitochondria. The P/O values of wild-type and mutant mitochondria were 1.31 and 1.17, respectively, with succinate as the substrate. Partial restoration of respiration in shy1 revertants is explained by an increase in COX The mitochondrial concentration of cytochromes a and a3 is a reliable gauge of COX content and activity. Spectra of mitochondrial cytochromes indicated the revertants to have more ‘a’ type cytochromes than the mutant (Figure 2B). The increase in spectrally detectable cytochrome oxidase was also confirmed by direct enzyme assays. The COX activities of mutant and revertant mitochondria were assayed spectrophotometrically by measuring oxidation of ferrocytochrome c, and polarographically by monitoring oxygen consumption with ascorbate plus N, N, N′,N′-tetramethyl-p-phenylenediamine (TMPD) as the substrate (Figure 2A). To maximize substrate availability in the spectrophotometric assay, mitochondria were permeabilized with low concentrations of deoxycholate. Under all assay conditions, the specific activities of COX in revertant mitochondria were approximately four times higher than in the mutant and ranged from 37 to 40% of wild type. Consistent with the above results, western blot analysis of total mitochondrial proteins revealed a higher steady-state concentration of COX subunits in the revertant than in the shy1 null mutant (Figure 2C). This is not due to any effect of the suppressor on transcription or processing of the mitochondrially derived mRNAs (data not shown). Subunit 1 (Cox1), subunit 2 (Cox2) and subunit 3 (Cox3) were ascertained to be correctly inserted in the inner mitochondrial membrane, as demonstrated by their susceptibility to proteinase K digestion in [35S]methionine-labeled mitoplasts (data not shown). In such assays, proteins that are not inserted into the phospholipid bilayer of the inner membrane are protected against proteinase K due to their location on the matrix side of the membrane (Hell et al., 2000). Identification of MSS51 as the suppressor The extragenic suppressor in ΔSHY1/R1 was cloned by transformation of the shy1 null mutant with a genomic library constructed from the nuclear DNA of the revertant. Transformation of ΔSHY1 with the library yielded ∼70 000 clones, of which five were respiratory competent. Plasmids obtained from the respiratory-competent transformants were amplified in Escherichia coli and their nuclear DNA inserts characterized. Restriction mapping indicated all five plasmids to have identical or overlapping fragments of nuclear DNA. The sequences of the end points of the insert in one of the plasmids (pSG91/T1) localized it between coordinates 550118 and 558028 on chromosome XII (Figure 3A). This plasmid was used to subclone the suppressor by transferring different regions of the insert to the yeast integrative vector YIp351 (Hill et al., 1986) and testing the ability of the new constructs to confer respiration on ΔSHY1 (Figure 3A). The results of these transformations indicated that the region of DNA containing only MSS51 was sufficient to produce the revertant phenotype. The identification of MSS51 as the suppressor was confirmed by linkage analysis. Diploid cells issued from a cross of ΔSHY1/R1 to the mss51 point mutant E4-218 were sporulated and 14 tetrads were dissected. In every case, only two of the meiotic spore progeny were respiratory competent, indicating linkage of the suppressor to MSS51. Figure 3.Identification of MSS51 as the suppressor gene. (A) Restriction maps of pSG91/T1 and of subclones. The locations of the restriction sites for BamHI (B), EcoRI (E), BglII (G), HindIII (H), KpnI (K) and SphI (Sp) are shown above the nuclear DNA insert in pSG91/T1. The regions of the nuclear insert in pSG91/T1 subcloned in YIp351 are represented by the solid bars in the upper part of the figure. The discontinuous lines represent the regions of pSG91/T1 deleted in each subclone. The plus and minus signs indicate suppression or lack thereof, respectively, of the shy1 null mutant by the subclones. The MSS51 reading frame and the direction of transcription of the gene are indicated by the solid arrow. The direction of transcription of the adjoining QRI5 gene is shown by the open arrow for orientation purposes. (B) Hydropathy profile of Mss51p (Kyte and Doolittle, 1982). The arrows indicate the location of the suppressor mutations identified. Download figure Download PowerPoint The sequence of MSS51 in pSG91/T1 disclosed a T595A transversion resulting in an F199I change in a hydrophobic region of the protein (Figure 3B). A second mutation in this region was identified in ΔSHY1/R2. The mutation in the latter revertant is a C500G base change resulting in the substitution of an arginine for a threonine at residue 162. The mss51F199I and mss51T167R alleles do not affect growth of an otherwise wild-type strain on non-fermentable substrates either at 30 or 37°C (data not shown). Suppression of the ΔSHY1 by both alleles, however, is temperature sensitive and is almost completely abolished at 37°C (Figure 1C). The high frequency with which shy1 null mutants revert could indicate the existence of multiple suppressor genes. Alternatively, suppression activity could be conferred by different mutations in MSS51. Genetic analyses of seven revertants indicated that in all cases the suppressors were linked to MSS51. The revertants used in the linkage analysis included two revertants of an ‘a’ mating type ΔSHY1 mutant, three revertants of an α mating type ΔSHY1 mutant, and two revertants of the shy1 point mutant W125. The lower yield of revertants at 37°C (see Figure 1A) suggested that they might represent a different class of mutations. This turned out not to be the case, as mutations in two revertants isolated at the higher temperature were also linked to MSS51. Suppression of shy1 mutants by wild-type MSS51 Wild-type MSS51 functions as a suppressor of shy1 mutants when present in two or more copies. Integration of MSS51 at the chromosomal LEU2 locus of shy1 mutants restores growth on rich glycerol/ethanol medium, although suppression is not as effective as the suppressor alleles (compare ΔSHY1/ST9 to ΔSHY1ΔMSS51/T5 or T7 in Figure 4). As expected, integration of the wild-type construct (pG91/ST9) in a mutant deleted for both SHY1 and MSS51 failed to restore respiratory growth (ΔSHY1ΔMSS51/ST9 in Figure 4). It is noteworthy that increasing the number of copies of MSS51 above two did not further improve the mutant's ability to respire (data not shown). Figure 4.Suppression of the respiratory defect of shy1 mutants by wild-type and mutant MSS51. MSS51 was cloned in YIp351 and integrated at the chromosomal LEU2 locus of the shy1 null mutant. MSS51 and the two suppressor genes mss51T167R and mss51? were also integrated at the LEU2 locus of ΔSHY1ΔMSS51, a mutant construct with null mutations in MSS51 and SHY1. Serial dilution of the wild-type W303-1A, the single and double mutants and the transformants starting with 105 cells were spotted on rich glucose (YPD) and rich glycerol/ethanol (YEPG) plates and incubated at 30 and 37°C for 2.5 days. Download figure Download PowerPoint Analysis of Cox1 in shy1 mutants and revertants Mss51p is a specific translation factor for COX1 mRNA (Faye and Simon, 1983; Siep et al., 2000). Although mss51 mutants are blocked in processing of the COX1 pre-mRNA, this is probably secondary to the translation defect. The ability of wild-type and mutant forms of MSS51 to partially correct the COX deficiency of shy1 mutants suggested that the mechanism of suppression might, in some way, be related to expression of the mitochondrial COX1 gene. The effect of shy1 mutations on translation and/or stability of Cox1 were examined by pulse labeling of whole cells and isolated mitochondria with [35S]methionine. In both assays, Cox1 was visibly less labeled in the mutant than in wild type or the revertant (Figure 5A and B). The incorporation of [35S]methionine into the different mitochondrial translation products was quantitated in several independent in vivo and in organello experiments. The results, summarized in Table I, indicate that the label in Cox1, when normalized to cytochrome b, Cox2 (subunit 2) or Cox3 (subunit 3), is 3–4 times lower in the mutant than the wild type. Cox1 in the revertant was as well labeled as in wild type, even though the ratios reported in Table I are somewhat lower in the revertant because of a greater incorporation of [35S]methionine into the other subunits. Figure 5.Mitochondrial protein synthesis in shy1 null mutants and revertants. (A) In vivo labeling of mitochondrial DNA products. Wild-type (W303), mutant (ΔSHY1) and revertant (ΔSHY1/R1) cells were labeled with [35S]methionine at 30°C for the times indicated in the presence of cycloheximide. (B) In organello protein synthesis. Mitochondria isolated from the same strains were labeled with [35S]methionine at 30°C in the absence of cycloheximide. Equivalent amounts of total cellular or mitochondrial proteins were separated by PAGE on a 17.5% polyacrylamide gel, transferred to a nitrocellulose membrane and exposed to an X-ray film. The mitochondrial translation products are identified on the left. The migration of Cox2 relative to Cox1 differs from that seen in (A) because a different PAGE buffer system was used for the separation. Download figure Download PowerPoint Table 1. Cox1p expression in wild-type and shy1 mutants Ratios Exp. 1 Exp. 2 Exp. 3 WT ΔSHY1 ΔSHY1/R1 WT ΔSHY1 ΔSHY1/R1 WT ΔSHY1 ΔSH1/R1 In organello Cox1/Cox2 4.43 1.55 3.78 4.57 1.28 3.98 – – – Cox1/Cox3 3.05 0.33 2.40 3.11 0.42 2.28 1.30 0.36 1.30 Cox1/cyt b 9.59 1.43 6.29 6.82 1.15 5.86 3.90 0.60 2.80 In vivo Cox1/Cox2 1.46 0.55 1.27 0.93 0.46 1.13 1.27 0.27 0.96 Cox1/Cox3 1.60 0.57 1.03 0.95 0.56 1.17 1.42 0.40 1.18 Cox1/cyt b 1.74 0.42 1.48 2.66 0.48 2.05 1.95 0.44 1.69 Mitochondrial translation products were labeled with [35S]methionine for 20 min at 23°C in organello and in vivo in the presence of cycloheximide as described in Materials and methods. Proteins were separated by PAGE on a 17.5% polyacrylamide gel and then transferred to a nitrocellulose membrane. The radioactivity associated with Cox1, Cox2, Cox3 and cytochrome b was quantitated in a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The table reports the results of three independent experiments. Different preparations of mitochondria were labeled in each of the in organello experiments. Most mitochondrial translation products, including Cox1, were maximally labeled under in vivo conditions after a 10 min pulse (Figure 5). To assess better the effect of the shy1 mutation on the amount of Cox1 available for assembly, the rate of incorporation of [35S]methionine into the different translation products was also measured at shorter times. The results of such experiments indicate that incorporation of [35S]methionine into Cox1 in the mutant is <20% of wild type, even at the earliest time points (Figure 6). This was not true of Cox2 or cytochrome b, both of which were labeled to approximately the same extent in the mutant and wild type. Although there was also less labeling of Cox3 in the mutant, the effect was not nearly as dramatic (<50% reduction) as with Cox1. Cox1 was equally well labeled in the revertant and wild-type cells during short pulses, as was the case when translation was allowed to proceed for longer times (Figure 5). Cox2 and Cox3 were also more labeled in the revertant than in wild type (Figure 6). Figure 6.Kinetics of in vivo labeling of mitochondrial products during short pulses. (A) Wild-type (W303), mutant (ΔSHY1) and revertant (ΔSHY1/R1) cells were labeled with [35S]methionine. Total cellular proteins were extracted, depolymerized in sample buffer and separated on a 17.5% polyacrylamide gel. The labeling conditions, sample preparation and gel electrophoresis were identical to those described in Figure 5A. The proteins were transferred to nitrocellulose and exposed to X-ray film. (B) The radioactivity associated with Cox1, Cox2 and Cox3, and with cytochrome b was also quantitated in a PhosphorImager. The results were analyzed by linear regression. Download figure Download PowerPoint The poor labeling of Cox1 in the mutant could be due to an effect of the shy1 mutation on the rate of synthesis and/or turnover of the protein. A direct requirement of Shy1p for translation of Cox1 is unlikely in view of the rates measured in cells that had been allowed to incubate for 2 h in medium containing chloramphenicol prior to the in vivo assay. The incubation in chloramphenicol leads to an accumulation of cytoplasmically synthesized proteins that act to increase the translation rate, presumably by drawing newly synthesized mitochondrial products into their respective assembly pathways (Tzagoloff, 1971). In agreement with earlier studies, the chloramphenicol pre-incubation stimulated [35S]methionine incorporation into most mitochondrial products in the wild-type strain, including Cox3, cytochrome b and Atp6 (Figure 7A). This was also true of the mutant. In addition, the chloramphenicol pre-incubation doubled the incorporation of label into Cox1 in the mutant, but not in the wild type (Figure 7A and B). Similar results were obtained with the point mutant W125. Figure 7.Effect of chloramphenicol on the kinetics of in vivo labeling of mitochondrial products. (A) Cells were grown and labeled at 30°C as in Figure 6, except that one half of the culture was incubated in the presence of 2 mg/ml chloramphenicol during the last 2 h of growth (+ CAP). Cells were harvested from both media and washed twice with a solution containing 40 mM potassium phosphate plus 2% galactose prior to labeling. Samples were removed after the labeling times indicated. Total cellular proteins were separated on a 17.5% gel as in Figure 6. (B) The radiolabeled Cox1 was quantitated in a PhosphorImager and the results were analyzed by linear regression. Download figure Download PowerPoint Turnover of mitochondrial translation products The results of the previous section suggested that the Cox1 deficit in shy1 mutants is not due to a direct role of Shy1p in translation, although an indirect effect on translation was not excluded. The deficit of Cox1 could be the result of increased turnover of Cox1. The stability of newly translated mitochondrial products was examined by pulse–chase experiments. Cells were pulsed with [35S]methionine at 30°C for 20 min in the presence of cycloheximide, and chased at the same temperature for different times following addition of puromycin and excess cold methionine. The products made during the pulse were relatively stable in all three strains for the first 5 min of the chase, but at longer times were progressively degraded (Figure 8). The exception was cytochrome b, whose concentration did not change even after 90 min of chase. While there was some decrease of Cox1 in the wild type and the revertant, the residual 20% of Cox1 in the mutant did not decrease further during the chase. In other experiments we observed some decrease of subunit 1 in the mutant, but less than in the wild type and the revertant. The explanation for the apparent greater stability of unassembled subunit 1 in the mutant is not clear, but could be related to its lower concentration compared with wild type or the revertant. Figure 8.Turnover of in vivo labeled mitochondrial translation products. Cells were grown and labeled for 20 min at 30°C with [35S]methionine as in Figure 5. The labeling reaction was terminated by addition of excess 80 mM cold methionine and 4 μg/ml puromycin (0 time). Samples of the cultures were collected after the incubation times at 30°C indicated and processed as in Figure 6. Download figure Download PowerPoint Do Mss51p and Shy1p interact with each other and/or with COX subunits? Shy1p and Mss51p are located in the mitochondrial inner membrane. To test whether they exist in a complex, wild-type mitochondria were extracted either with deoxycholate or laurylmaltoside and the properties of the solubilized proteins were examined by sedimentation through sucrose gradients. The results of the sucrose gradient analysis indicated Shy1p and Mss51p to behave as distinct proteins. The mass of native Shy1p solubilized with deoxycholate was estimated to be 95 kDa, a value approximately two times higher than the mass of the monomer. Similarly, the value of 115 kDa obtained for the mass of native Mss51p is 2–3 times greater than that of the monomer. Each protein peaked in a separate fraction with a homogeneous distribution in the gradient (Figure 9). Identical values were obtained when Shy1p was extracted from mitochondria of a mss51 null mutant and conversely when Mss51p was extracted from a shy1 null mutant, confirming the absence of a stable complex of the two proteins. Figure 9.Sedimentation properties of Shy1p and Mss51p. Mitochondria (7 mg of protein) from the wild-type haploid strain W303-1A were solubilized in the presence of 1 M KCl and 1% KDOC. The clarified extract obtained by centrifugation at 200 000 gav for 15 min was mixed with hemoglobin and lactate dehydrogenase, and applied to 5 ml of a linear 10–25% sucrose gradient containing 10 mM Tris–HCl pH 7.5, 0.5 mM EDTA and 0.1% Triton X-100. Following centrifugation for 14 h at 50 000 r.p.m. in a Beckman SW65 rotor, the gradient was