Identification of amino acid residues of mammalian mitochondrial phosphate carrier important for its functional expression in yeast cells, as achieved by PCR-mediated random mutation and gap-repair cloning

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (Delta NrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, Delta NrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that Delta NrPiC expressed in yeast mitochondria is inert. Next we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated Delta NrPiC, and obtained two clones. These clones carried either of two mutations, F2675 or F282S; and these mutations restored the transport function of Delta NrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of Delta NhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria. (C) 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.