Two related genes encoding extremely hydrophobic proteins suppress a lethal mutation in the yeast mitochondrial processing enhancing protein.
The processing enhancing protein of mitochondria (PEP) is an essential component that has been shown to participate in proteolytic removal of NH2-terminal signal peptides from precursor proteins imported into the mitochondrial matrix. Using a yeast strain bearing a PEP mutation that renders it temperature-sensitive, an approach of genetic suppression was taken in order to identify additional components that could be involved with protein import: high copy plasmids comprising a yeast genomic library were tested for ability to suppress the 37 degrees C growth defect. Two plasmids were isolated, pSMF1 and pSMF2, which suppressed the growth defect nearly as well as the cloned PEP gene itself. Sequence analysis of the rescuing genes predicted extremely hydrophobic proteins with sizes of 63 and 60 kDa, respectively. Remarkably, the predicted SMF1 and SMF2 products are 49% identical to each other overall. To test the requirement for SMF1 and SMF2, the chromosomal genes were disrupted. Individual disruption was without effect, but cells in which both genes were disrupted grew poorly. When mitochondria were prepared from the double disruption strain grown in a nonfermentable carbon source, they were morphologically normal but defective for translocation of radiolabeled precursor proteins. SMF1 protein was provisionally localized to the mitochondrial membranes using epitope tagging. We suggest that SMF1 and SMF2 are mitochondrial membrane proteins that influence PEP-dependent protein import, possibly at the step of protein translocation.
West
A H
AH
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Clark
D J
DJ
Martin
J
J
Neupert
W
W
Hartl
F U
FU
Horwich
A L
AL
D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005804:Genes, Lethal; D016153:Genes, Suppressor; D005838:Genotype; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D013696:Temperature
D004271:DNA, Fungal; D010455:Peptides