Mutation of K234 and K236 in the voltage-dependent anion channel 1 impairs its insertion into the mitochondrial outer membrane.
Previous in vitro studies indicated that mutation of both K234 and K236 to arginine, glutamine, or glutamic acid impaired the ability of the voltage-dependent anion channel (VDAC1) to insert into the outer membrane of the mitochondria (Smith et al. 1995). These same mutants were expressed in a strain of Saccharomyces cerevisiae with a disruption in the VDAC1 gene. The mutant VDAC1 forms were found in the mitochondria suggesting that they were correctly sorted to the outer membrane. However, only very small amounts of the mutants were inserted into the mitochondrial membranes. Mitochondria isolated from the strains expressing the mutants were capable of catalyzing the translocation of both wild-type VDAC1 and pre-alcohol dehydrogenase III indicating that the translocation apparatus was functional. These results confirm the previously drawn conclusion that K234 and K236 are part of a membrane insertion motif. The failure of the mutant VDAC1 forms to insert did not cause VDAC1 precursors to accumulate in the soluble cell cytoplasm or in the microsomal fraction. The apparent lack of a "precursor pool" suggested that a post-transcriptional control mechanism might limit the amounts of VDAC1 precursors in the cell. Such a control mechanism is consistent with the observation that the amount of VDAC1 was very similar after epichromosomal (gene in a 2u plasmid controlled by a Gal1 promoter) and chromosomal expression (endogenous gene controlled by the endogenous promoter).
Angeles
R
R
Department of Pharmacology, School of Medicine, Wayne State School of Medicine, Detroit, Michigan 48201, USA.
Devine
J
J
Barton
K
K
Smith
M
M
McCauley
R
R
D019943:Amino Acid Substitution; D015825:Chromosomes, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis; D007425:Intracellular Membranes / Q000502:physiology*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D013104:Spheroplasts / Q000502:physiology; D050995:Voltage-Dependent Anion Channel 1; D050994:Voltage-Dependent Anion Channels
D005656:Fungal Proteins; D018272:Porins; D011994:Recombinant Proteins; D050994:Voltage-Dependent Anion Channels; D050995:Voltage-Dependent Anion Channel 1; D007930:Leucine