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ID 113788
Author
Yamagoshi, Ryohei Tokushima University
Hashimoto, Mitsuru Matsuyama University
Sugahara, Ryohei National Institute of Agrobiological Sciences
Shiotsuki, Takahiro National Institute of Agrobiological Sciences
Miyoshi, Hideto Kyoto University
Terada, Hiroshi Niigata University of Pharmacy and Applied Life Sciences
Keywords
Mitochondrial solute carrier
Phosphate carrier
Functional expression
Gap-repair cloning
Yeast
Content Type
Journal Article
Description
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.
Journal Title
Mitochondrion
ISSN
15677249
NCID
AA11694235
AA11643835
Publisher
Elsevier|Mitochondria Research Society
Volume
32
Start Page
1
End Page
9
Published Date
2016-11-09
Rights
© 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
FullText File
language
eng
TextVersion
Author
departments
Institute of Advanced Medical Sciences