Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae

Abstract

Expression of yeast mitochondrial genes depends on specific translational activators acting on the 5'-untranslated region of their target mRNAs. Mss51p is a translational factor for cytochrome c oxidase subunit 1 (COX1) mRNA and a key player in down-regulating Cox1p expression when subunits with which it normally interacts are not available. Mss51p probably acts on the 5'-untranslated region of COX1 mRNA to initiate translation and on the coding sequence itself to facilitate elongation. Mss51p binds newly synthesized Cox1p, an interaction that could be necessary for translation. To gain insight into the different roles of Mss51p on Cox1p biogenesis, we have analyzed the properties of a new mitochondrial protein, mp15, which is synthesized in mss51 mutants and in cytochrome oxidase mutants in which Cox1p translation is suppressed. The mp15 polypeptide is not detected in cox14 mutants that express Cox1p normally. We show that mp15 is a truncated translation product of COX1 mRNA whose synthesis requires the COX1 mRNA-specific translational activator Pet309p. These results support a key role for Mss51p in translationally regulating Cox1p synthesis by the status of cytochrome oxidase assembly.

Figure 1.

Pet309p and COX1-dependent synthesis of a novel protein in mss51 null mutants. (A) In vivo synthesis of mp15 by mss51 mutants requires the presence of Pet309p. Mitochondrial products of wild type (W303) and of pet309 and mss51 single or double mutants were labeled with [³⁵S]methionine at 30°C for 10 min in the presence of cycloheximide (Barrientos et al., 2002b). (B) A partial deletion of COX1 blocks in vivo synthesis of mp15. Mitochondrial products were labeled as in A in the wild type W303-1A (W303) and mss51 null mutants (Δmss51) in different mitochondrial genetic backgrounds: W303-mtDNA (ρ^W303), D273-mtDNA (W303ρ^D273), Δmss51ρ^D273), or Δcox1-mtDNA (Tzagoloff et al., 1975), the later a D273 type of D273-mtDNA with a partial deletion of the COX1 gene obtained from strain M5.16-A3 (Table 1). Δmss51r^Dcox1 -1 and -2 and Δmss51ρ^D273 -1 and -2 are two different cytoductants of each type.

Figure 2.

Electrophoretic mobility of mp15. Mitochondrial products were labeled in vivo in a wild type (W303-1A) and a mutant strain carrying a null allele of mss51 (Δmss51) as in Figure 1. Total cellular proteins were separated in a 17.5% PAGE (Barrientos et al., 2002b). Cox1p of the wild-type cells and mp15 from the mss51 mutant were excised from the SDS polyacrylamide gel and electroeluted using ElutaTube Protein Extraction kit (Fermentas, Ontario, Canada). The proteins were trichloroacetic acid (TCA) precipitated and resuspended in loading buffer. The radiochemical purity of the two proteins was tested by separation on a second 12% polyacrylamide gel (Laemmli, 1970) or a 20% polyacrylamide gel in Tris-Tricine buffer (Schagger and von Jagow, 1987).

Figure 3.

Accumulates of mp15 in vivo in COX assembly-arrested mutants. (A) Wild type (W303-1A), mss51, cox14, cox16, cox18, sco1, cox7, and cox17 single null mutants or double mutants with a second null mutation in PET309 were labeled with [³⁵S]methionine at 30°C for 15 min in the presence of cycloheximide as in Figure 1. (B) Wild type (W303-1A), cox14 single mutant or double mutants with a second mutation in shy1, cox15, and sco1 were labeled with [³⁵S]methionine at 30°C for 15 min in the presence of cycloheximide as in Figure 1. The mitochondrial translation products are identified in the margin. The functions affected in the different strains are described in Table 1.

Figure 4.

COX14 does not affect synthesis and accumulation of mp15. (A) Wild type (W303-1A), an mss51 null mutant, and an mss5/cox14 double mutant were labeled with [³⁵S]methionine at 30°C for 15 min in the presence of cycloheximide as described in Figure 1. (B) Wild type (W303-1A), and a null mutant of mss51 overexpressing or not COX14, were labeled in vivo as described in A. The mitochondrial translation products are identified in the margin.

Figure 5.

Synthesis and/or accumulation of mp15 are reduced in COX assembly mutants pretreated with chloramphenicol. (A) Effect of CAP pretreatment on in vivo labeling of mitochondrial gene products. Wild type (W303-1A) and the mss1 null mutant (Δmss51) were grown in YPGal. One-half of the cultures were further incubated at 30°C for 2 h in the presence of 2 mg/ml CAP. Cells were harvested and washed two times with a solution containing 40 mM potassium phosphate plus 2% galactose before labeling. Samples were removed after the indicated times of labeling and processed as in Figure 1. (B) Degradation of mp15 synthesized in cells pretreated or not with CAP. The mss1 null mutant (Δmss51) was incubated in the presence and absence of CAP as described in A. After harvesting and washing, cells were pulsed with [³⁵S]methionine for 10 min. Labeling was terminated by addition of 80 μmol of cold methionine and 12 μg/ml puromycin (0 time). Samples of the cultures were collected after the indicated times of incubation at 30°C and processed as described in Figure 1. (C) Quantification of mp15 degradation in B. The radiolabeled bands were detected and quantified with a PhosphorImager (GE Healthcare). The values (arbitrary units) were plotted against the time of chase. (D) Effect of CAP pretreatment on the mitochondrial protein synthesis pattern of cox10, imp1, and cox7 null mutants. Cells were labeled as in A. Mitochondrial translation products are identified in the margin. Cox2p is not processed in Δimp1 mutant. The Cox2p precursor (pCox2p) in these strain migrates slower that the mature Cox2p (mCox2p). The functions affected in the different mutants are described in Table 1.

Figure 6.

mp15 is a membrane protein whose degradation depends partially on a proteolytically active YTA10/YTA12 complex. (A) mp15 is a membrane protein. Wild type (W303-1A) and a mutant carrying a null allele of mss51 were grown and labeled for 15 min at 30°C with [³⁵S]methionine in the presence of cycloheximide. Cells were subsequently converted to spheroplasts by digestion of the cell wall with zymolyase and submitted to sonic radiation. Samples were centrifuged 25,000 rpm, and the pellet (P) and supernatant (S) fractions were collected, TCA precipitated, and resuspended in loading buffer. The proteins were separated into a 17.5% PAGE and transferred to a nitrocellulose membrane. The membrane was both, exposed to x-ray film (bottom) and used for a Western blot that was probed with a polyclonal antibody against the matrix soluble protein α-ketoglutarate dehydrogenase (top). (B) mp15 is rapidly degraded after synthesis. Wild type (W303-1A) and a mutant carrying a null allele of mss51 were grown and labeled for 15 min at 30°C with [³⁵S]methionine. Labeling was terminated by addition of 80 μmol of cold methionine and 12 μg/ml puromycin (0 time). Samples of the cultures were collected after the indicated times of incubation at 30°C and processed as in Figure 1. (C) Degradation of mp15 is partially mediated by the YTA10/YTA12 complex. The wild type strain W303-1A, the shy1, and cox11 mutants, and the same mutants in which the endogenous wild-type YTA10 gene had been substituted by the catalytically inactive yta10^E559Q mutant gene (Arlt et al., 1996) were labeled and chased for the indicated times as in A. Mitochondrial translation products are identified in the margin.

Figure 7.

Synthesis of mp15 depends on the intron composition of the COX1 gene. The figure represents the maps of COX1 in different strains of yeast. Group II introns are circled. mtDNA containing COX1 genes with different intron compositions (see details in Table 1) were transferred by cytoduction into to a kar1 mutant (Conde and Fink, 1976) devoid of mtDNA (ρ^o). The different mitochondrial genomes were transferred from the kar1 donor to ρ^o derivatives of mss51 and shy1 null mutants. To determine whether mp15 was synthesized in these strains, the mitochondrial gene products of the corresponding strains were labeled in vivo in the presence of cycloheximide and separated on a 17.5% polyacrylamide gel as in Figure 1. The last two columns on the right represent a summary of the results obtained.

Figure 8.

Mature and unprocessed COX1 transcripts accumulate in mss51 and shy1 but not in pet309 null mutants. (A) Mitochondrial RNA was extracted from mitochondria purified of the wild-type strain W303-1A, a shy1 null mutant (Δshy1), an mss51 null mutant (Δmss51) and a double shy/1mss51 mutant (Δshy1 Δmss51). The RNA extracts were separated onto a 1% agarose gel, stained with ethidium bromide, photographed, and the RNAs blotted to a nylon membrane (Nytran, SuPerCharge; Whatman Schleicher and Schuell, Keene, NH). After cross-linking with UV light, the nylon membrane was prehybridized at 43°C with 125 μg of salmon sperm DNA in 5× SSC, 5× Denhardts, 0.5% SDS. The blotted RNAs were hybridized overnight at 43°C with probes containing exon 5 + intron 5 of COX1 and exon 1 of COB. Both probes were labeled with [α-³²P]dATP by random priming (Feinberg and Vogelstein, 1983). (B) Mitochondrial RNA extracts were separated onto a denaturing 1.2% agarose gel, blotted to a nylon membrane and UV-light cross-linked. Northern blots of mitochondrial RNA of the wild-type W303-1A, the mss51, pet309, and shy1 null mutants with a W303 mtDNA background (Figure 7A) and the mss51 null mutant with an intronless mitochondrial genome were prehybridized at 42°C for 2 h with 1 μg of salmon sperm and hybridized overnight at 65°C in a solution containing 7% SDS, 1 μM EDTA, and 0.5 M Na₂PO₄ with probes containing the entire coding sequence of COX1 or COB. The mature (M) and unprocessed (P) COX1 transcripts are identified in the margins. The left panels in A–C shows the ethidium bromide-stained gels used for the Northern blots. The positions of the 15S and 21S mitochondrial rRNAs are indicated.

Figure 9.

Mss51p binds to the 5′-UTR of COX1 mRNA. (A) Y3H strategy used to explore the interaction of Mss51p with the 5′-UTR of COX1 mRNA. (B) Diagram showing the regions of Mss51p (including a hydrophobicity map) and the COX1 mRNA that were used to make the Y3H constructs. (C) A domain in the N′-terminus of Mss51p (Mss51.140; marked with a square in the hydrophobicity map in B) interacts with a domain in the 5′-UTR of COX1 mRNA (Cox1.4; marked with an arrow in B). Activation of the β-galactosidase reporter gene detected as a blue color occurred only when both the protein prey and RNA bait were expressed.

Figure 10.

Model depicting the hypothetical roles of Mss51p in Cox1p translation and coupling to Shy1p-dependent COX assembly. Mss51p is required for translation of Cox1p by acting, together with Pet309p on the 5′-UTR of COX1 mRNA. As shown before (Barrientos et al., 2004), Mss51p binds newly synthesized Cox1p forming a transient complex that is stabilized by Cox14p. A downstream event, maybe catalyzed by Shy1p, causing Cox1p to dissociate from the ternary complex makes Mss51p available for new rounds of translation. To simplify the model we have not included the action of Mss51p on the coding sequence of COX1, probably on Cox1p itself, interpreted as necessary for elongation of the nascent polypeptide (Perez-Martinez et al., 2003). In mss51 mutants (Δmss51) Mss51p-independent translation from alternate initiation sites in a COX1 mRNA precursor occur generating a new polypeptide, mp15, which is proteolytically degraded.