Relocation of the unusual VAR1 gene from the mitochondrion to the nucleus.
Relocation of the unusual VAR1 gene from the mitochondrion to the nucleus.
复制标题
不寻常的 VAR1 基因从线粒体重新定位到细胞核。
DOI：
10.1139/o95-106
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Mason,TL
中科院分区：
文献类型：
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作者：
Sanchirico,M;Tzellas,A;Fox,TD;Conrad-Webb,H;Periman,PS;Mason,TL
Varl蛋白（Var1p）是酵母线粒体小核糖体亚基的重要化学计量成分，也是线粒体遗传系统中唯一不属于内膜能量转导复合体的主要蛋白质产物。有趣的是，没有突变被报道影响Var1p的功能，可能是因为线粒体翻译系统功能的丧失导致mtDNA的不稳定。为了研究Varlp的结构、功能和合成，我们利用细胞核基因VAR1U，将39个非标准线粒体密码子转化为通用密码子，设计酵母株表达该蛋白。使用表位标记形式的Var1Up进行免疫印迹分析显示，核编码蛋白被表达并导入线粒体。我们测试了var1u对mtDNA PZ206突变的补充能力，PZ206突变破坏了var1mrna的3 '端加工，导致Var1p的合成大大减少和呼吸缺陷表型。在adh1启动子控制下，PZ206突变体通过携带var1的着丝粒质粒转化恢复了呼吸生长，从而证明了var1功能可以从线粒体转移到细胞核。此外，表位标记的Var1Up在高盐蔗糖梯度下特异性地与小核糖体亚基共同沉积。Var1从线粒体到细胞核的重新定位为不寻常的Var1蛋白的结构-功能关系的分子遗传学分析提供了一个很好的系统。关键词：酿酒酵母，var1基因，线粒体，核糖体组装，基因重新定位，RNA加工，核-线粒体相互作用
The Varl protein (Var1p) is an essential, stoichiometric component of the yeast mitochondrial small ribosomal subunit, and it is the only major protein product of the mitochondrial genetic system that is not part of an energy transducing complex of the inner membrane. Interestingly, no mutations have been reported that affect the function of Var1p, presumably because loss of a functional mitochondrial translation system leads to an instability of mtDNA. To study the structure, function and synthesis of Varlp, we have engineered yeast strains for the expression of this protein from a nuclear gene,VAR1U, in which 39 nonstandard mitochondrial codons were converted to the universal code. Immunoblot analysis using an epitope-tagged form of Var1Up showed that the nuclear-encoded protein was expressed and imported into the mitochondria.VAR1Uwas tested for its ability to complement a mutation in mtDNA, PZ206, which disrupts 3′-end processing of theVAR1mRNA, causing greatly reduced synthesis of Var1p and a respiratory-deficient phenotype. Respiratory growth was restored in PZ206 mutants by transformation with a centromere plasmid carryingVAR1UunderADH1promoter control, thus proving thatVAR1function can be relocated from the mitochondrion to the nucleus. Moreover, epitope-tagged Var1Up co-sedimented specifically with small ribosomal subunits in high salt sucrose gradients. The relocation ofVAR1from the mitochondrion to the nucleus provides an excellent system for the molecular genetic analysis of structure–function relationships in the unusual Var1 protein.Key words:Saccharomyces cerevisiae,VAR1gene, mitochondria, ribosome assembly, gene relocation, RNA processing, nuclear–mitochondrial interaction.