哺乳动物的线粒体磷酸盐载体(PiC),但不是酵母,是用预排序合成的。据报道,哺乳动物PiC的这种前序列的缺失有助于将载体导入酵母线粒体,但是关于哺乳动物PiC是否可以在酵母线粒体中功能性表达的问题尚未解决。在本研究中,我们首先检查了是否可以通过引入大鼠PiC的表达载体来逆转缺乏酵母PiC基因的酵母细胞在甘油板上的生长缺陷。引入编码全长大鼠PiC(rPiC)或缺乏前序列的rPiC(ΔNrPiC)的表达载体对恢复甘油板上的生长无效。当我们检查酵母线粒体中单个rPiCs的表达水平时,ΔNrPiC的表达水平与酵母PiC相似,但rPiC很低.这些结果表明在酵母线粒体中表达的ΔNrPiC是惰性的。接下来,我们试图通过表达随机突变的ΔNrPiC来分离甘油板上的“回复体”,并获得了两个克隆。这些克隆携带两种突变中的一种,F267S或F282S;这些突变恢复了酵母线粒体中ΔNrPiC的转运功能。这两个Phe残基在人类携带者(hPiC)中保守,酵母线粒体中表达的ΔNhPiC的转运功能也通过它们的取代而显着改善。因此,F267S或F282S的取代被认为对于酵母线粒体中哺乳动物PiC的功能表达是重要的。
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.