Two nuclear mutations that block mitochondrial protein import in yeast.

Abstract

We isolated two yeast mutants that are temperature-sensitive for import of mitochondrial proteins. Each strain contains a single mutation that results in arrest of growth and accumulation of precursor to the f8 subunit of the mitochondrial Fl-ATPase after incubation at 37°C. These lesions (masi and mas2) are nonallelic and recessive. Cells harboring either mutation stop growing only after 2-3 generations at 37°C. Import of the F1 fl subunit at 37C is more than 250 times slower in masl and 15 times slower in mas2 than in wildtype cells. At 23°C, import occurs with si~ihar rates in mutant and wild-type cells. The two mutations also reduce the rate of import of other proteins; however, import of different precursors is affected to different degrees in the two strains. The temperature-sensitive step in import in both masi and mas2 occurs before arrival of precursors in the mitochondrial matrix.A central feature of mitochondrial assembly is the import of mitochondrial proteins from their site of synthesis in the extramitochondrial cytoplasm (1). Key steps in this import process have been identified by isotope-tracer studies with intact cells and by incubation of isolated mitochondria or mitochondrial subfractions with radiolabeled protein precursors synthesized in vitro (2, 3). These approaches have revealed that most mitochondrial proteins are synthesized as precursors of larger molecular weight (2, 4) and that import involves: (i) specific interaction of precursors with the mitochondrial outer membrane (5, 6), (ii) energy-dependent translocation of polypeptides across one or both mitochondrial membranes (7,8), and (iii) processing of the precursors by a matrix-localized protease (9). Thus far, little is known about how this import pathway is regulated, and most of its molecular components remain to be identified or isolated.Conditional yeast mutants have been an important tool for the dissection of many complex cellular phenomena such as the cell cycle (10) days at 23TC. Tdmperature-sensitive mutants were identified by replica-plating the colonies onto two sets of YPD-agar plates and incubating these at 370C and 23TC, respectively. After 2-3 days, a second replica was prepared from the 370C plate, and the replica was again incubated at 37°C. Strains which grew on the 230C plate but not on the second 37°C replica were scored as temperature-sensitive mutants. Import mutants were identified by screening temperaturesensitive mutants for accumulation of the precursor to the 83 subunit of the Fl-ATPase after incubation at 37°C. Cells were picked from agar slants to 250 ,l of YPD-10 medium in 2.2-ml Eppendorf tubes and grown overnight with shaking at 23°C. One milliliter of YPD medium was then added, and the cultures were incubated for 7 hr at 37°C. Cells were lysed by addition of 160 ,ul of fresh 1.85 M NaOH/7.4% 2-mercaptoethanol to the total culture. This mixture was held on ice for 10 min, and protein was then precipitated by addition of 160 Al of 50% (wt/vol) trichloroacetic acid. After a further 10 min on i...

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

“…Furthermore, the protein sequence of PEP shows 60% homology to that of the mas-7 gene product (Witte et al, 1988). This observation suggests that mas-7 encodes the yeast equivalent to the Neurospora PEP Interestingly, mas-7 cells at the nonpermissive temperature have been reported to accumulate precursors outside mitochondria (Yaffe and Schatz, 1984). This may support the idea of a role of PEP in the translocation of precursors into the mitochondria, as discussed above, in addition to its role in enhancing precursor processing.…”

“…Recently, temperature-sensitive mutations in Saccharomyces cereviseae were described that affect the processing of mitochondrial precursor proteins (Yaffe and Schatz, 1984;Yaffe et al, 1985). Two complementation groups were identified (mas-7 and mas-2) in which precursor proteins accumulated at the restrictive temperature.…”

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

“…A second interaction site at Arg 78 to Ser 91 turned out to be unspecific because it was not only also observed with NAC-(␤NAC⌬1-11) but also with the antibody control, suggesting that the positive signal at these spots was due to an epitope-independent unspecific binding of the antibody used for detection. When we searched for the localization of the peptide Val 23 to Lys 41 in Rpl31 in the context of the yeast ribosome we found that it is surface accessible and therefore a likely candidate for interaction with other proteins (see supplemental Fig. S5c).…”

Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast

“…Characterization of the conditional mutant GAL::bms1+ A: Scheme of the GAL::bms1 allele+ The BMS1 ORF is fused to ubiquitin, HA-tag, and LacI regions+ Following removal of ubiquitin, the fusion protein starts with the destabilizing amino acid, ariginine (R)+ B: Growth curves of the GAL::bms1 and WT strains in YPGal and YPD6%+ C: Expression of HA-Bms1p in the GAL::bms1 strain grown in YPGal (lane 2) or transferred to YPD6% for 3 and 6 h (lanes 3 and 4)+ Lane 1: extract from WT yeast; lane 5: extract from the BMS-HA strain, in which the C-terminally HA-tagged Bms1p (Bms1p-HA) is expressed from the chromosome under control of its own promoter+ Cell extracts were prepared in denaturing conditions and proteins were subsequently precipitated by trichloroacetic acid according to Yaffe and Schatz (1984) Bms1p is localized in the nucleolus+ Bms1p-HA expressed in the BMS1-HA strain was detected by a rat 3F10 a-HA Ab followed by a goat FITC-coupled a-rat Ab+ Nop1p was detected by mouse mAb A66 followed by a goat TRconjugated a-mouse Ab+ Chromatin was stained with DAPI+ Merging of all three images is shown in the bottom right panel+ 2000), was only barely visible, suggesting that processing at A 0 is strongly inhibited+ (2) As expected, a direct cleavage of the 35S pre-rRNA at A 3 resulted in the disappearance of the precursor 27SA 2 in cells depleted of Bms1p (probe c), whereas the precursor 27SA/B, extending downstream from site A 3 , was not affected (probe e)+ (3) Levels of 18S rRNA (probe g) and its immediate precursor 20S (probe b) were significantly decreased, whereas the level of mature 25S rRNA (probe f) was not changed in response to depletion of Bms1p+ To further investigate the processing defects at sites A 0 -A 2 , a primer extension analysis was performed (Fig+ 5B, panel h)+ It revealed strongly decreased levels of cDNA extending to site A 2 , consistent with the accumulation of 23S and the disappearance of 27SA 2 RNAs, as identified by northerns+ The amount of cDNA extending to site A 0 was not increased upon Bms1p depletion+ This contrasts with the situation observed in cells deficient in Rcl1p and is consistent with very low levels of the 22S RNA detected on northern blots+…”