The MAS-encoded processing protease of yeast mitochondria. Overproduction and characterization of its two nonidentical subunits.
The amino-terminal presequences of proteins imported from the cytoplasm across the mitochondrial inner membrane are cleaved off by a soluble matrix-localized protease composed of two nonidentical homologous subunits. In the yeast Saccharomyces cerevisiae, these are encoded by the nuclear MAS1 and MAS2 genes. We have now constructed yeast strains in which either one or both of the genomic MAS genes are controlled by a galactose-inducible strong promoter. In these strains, the intramitochondrial concentration of each MAS-encoded subunit as well as of the holo-protease can be varied over a wide range. When overproduced, the MAS1 protein precipitates in the matrix whereas the MAS2 protein remains soluble. The MAS2 protein was obtained at a purity of 98% in milligram amounts. The purified MAS2 subunit exists largely as a soluble 52-kDa monomer. Its cleavage activity is very low and might well reflect the 2% contamination by holoprotease. Activity is restored by adding the solubilized purified MAS1 subunit. Yeast cells depleted of one or both MAS subunits continue to import precursor proteins into mitochondria, but fail to cleave them; eventually the deficient cells stop growing. This growth arrest is partly suppressed on minimal medium or under conditions in which the cells are less dependent on mitochondrial metabolism. Depletion of the MAS1 subunit causes overproduction of the MAS2 subunit.
Geli
V
V
Biocenter, University of Basel, Switzerland.
Yang
M J
MJ
Suda
K
K
Lustig
A
A
Schatz
G
G
D002467:Cell Nucleus / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal; D005838:Genotype; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D046911:Macromolecular Substances; D010450:Endopeptidases