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 (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC 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.
Institute for Genome Research, Tokushima University, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, Tokushima University, Shomachi-1, Tokushima 770-8505, Japan.
Ryohei
R
Yamagoshi
Institute for Genome Research, Tokushima University, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, Tokushima University, Shomachi-1, Tokushima 770-8505, Japan.
Takenori
T
Yamamoto
Faculty of Pharmaceutical Science, Matsuyama University, Bunkyocho-4, Matsuyama 790-8578, Japan.
Mitsuru
M
Hashimoto
Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Ryohei
R
Sugahara
Insect Growth Regulation Research Unit, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Takahiro
T
Shiotsuki
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Hideto
H
Miyoshi
Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
Hiroshi
H
Terada
Institute for Genome Research, Tokushima University, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, Tokushima University, Shomachi-1, Tokushima 770-8505, Japan. Electronic address: yshinoha@genome.tokushima-u.ac.jp.
Yasuo
Y
Shinohara
D019943:Amino Acid Substitution; D000818:Animals; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004252:DNA Mutational Analysis; D015870:Gene Expression; D005990:Glycerol / Q000378:metabolism; D016296:Mutagenesis; D028061:Phosphate Transport Proteins / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D003470:Culture Media; D028061:Phosphate Transport Proteins; D011994:Recombinant Proteins; D005990:Glycerol
Functional expression; Gap-repair cloning; Mitochondrial solute carrier; Phosphate carrier; Yeast