ISOLATION OF YEAST MUTANTS DEFECTIVE IN PROTEIN TARGETING TO THE VACUOLE
ISOLATION OF YEAST MUTANTS DEFECTIVE IN PROTEIN TARGETING TO THE VACUOLE
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DOI：
10.1073/pnas.83.23.9075
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发表时间：
1986-12-01
影响因子：
11.1
通讯作者：
EMR, SD
中科院分区：
文献类型：
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作者：
BANKAITIS, VA;JOHNSON, LM;EMR, SD
我们构建了PRC 1-SUC 2基因融合体，该融合体指导酿酒酵母合成杂合多肽，该杂合多肽由源自酵母液泡蛋白酶羧肽酶Y的433个残基氨基末端结构域（CPY; EC 3.4.16.1）和源自分泌型酵母酶转化酶的511个残基羧基末端结构域（EC 3.2.1.26）。分级数据表明，该量的CPY一级序列足以定量地将转化酶转移到酵母液泡。将活性转化酶定位于液泡的表型结果使我们能够选择将杂交蛋白“错误定位”于细胞表面的突变体。导致这种效应的相应突变都是反式作用和隐性的，它们定义了至少八个互补组。这些空泡蛋白靶向（vpt）突变体也表现出杂合蛋白的非依赖性作用，在野生型CPY交付到酵母空泡。CPY的前体形式在突变体中积累并分泌到酵母周质和细胞外培养基中。vpt突变体应该提供有用的信息，酵母细胞调节液泡蛋白运输的机制。
We have constructed a PRC1-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that "mislocalize" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent effects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic.