Mutation of K234 and K236 in the voltage-dependent anion channel 1 impairsits 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-d ependent anion channel (VDAC1) to insert into the outer membrane of the mit ochondria (Smith et al. 1995). These same mutants were expressed in a strai n of Saccharomyces cerevisiae with a disruption in the VDAC1 gene. The muta nt VDAC I forms were found in the mitochondria suggesting that they were co rrectly sorted to the outer membrane. However, only very small amounts of t he mutants were inserted into the mitochondrial membranes. Mitochondria iso lated from the strains expressing the mutants were capable of catalyzing th e translocation of both wild-type VDAC1 and pre-alcohol dehydrogenase III i ndicating that the translocation apparatus was functional. These results co nfirm the previously drawn conclusion that K234 and K236 are part of a memb rane insertion motif. The failure of the mutant VDAC1 forms to insert did n ot 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 V DAC1 precursors in the cell. Such a control mechanism is consistent with th e observation that the amount of VDAC1 was very similar after epichromosoma l (gene in a 2u plasmid controlled by a Gall promoter) and chromosomal expr ession (endogenous gene controlled by the endogenous promoter).