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首页> 外文期刊>Molecular and Cellular Biology >Human ARF4 expression rescues sec7 mutant yeast cells.
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Human ARF4 expression rescues sec7 mutant yeast cells.

机译：人类ARF4表达可拯救sec7突变酵母细胞。

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Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.
机译：酵母分泌途径的区室之间的囊泡介导的运输涉及募集多种胞质蛋白以用于出芽，靶向和膜融合事件。 SEC7基因产物（Sec7p）是在运输途中到达啤酒酵母中的高尔基复合体的囊泡上的被膜结构的组成部分。为了鉴定Sec7p及其相互作用蛋白的哺乳动物同源物，我们使用了一种基因选择策略，其中将人类HepG2 cDNA文库转化为条件致死酵​​母sec7突变体。我们分离了几个能够在限制性温度下拯救sec7突变体生长的克隆。编码最有效抑制剂的cDNA被鉴定为人ADP核糖基化因子4（hARF4），它是GTPase家族的成员，被提议用来调节哺乳动物细胞中囊泡外壳蛋白的募集。已经确定了与Sec7p相互作用的蛋白，而不是哺乳动物的Sec7p同源物，我们提供的证据表明hARF4通过恢复分泌途径的功能抑制了sec7突变。将sec7菌株转移到限制性温度会导致突变的Sec7p胞质池消失，而膜相关部分没有明显变化。向细胞中引入hARF4可以维持细胞溶质和膜相关Sec7p库之间的平衡。这些结果表明，对于细胞生长和囊泡运输而言，Sec7p在膜上循环的必要性。此外，酵母GTPase编码基因ARF1和ARF2的过表达，而不是YPT1的过表达，以等位基因特异性的方式抑制了sec7突变体的生长表型。这种等位基因特异性表明，个体ARF被募集以在囊泡被膜动力学中执行两种不同的Sec7p相关功能。

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