Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases
Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases
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DOI：
10.1128/mcb.8.11.4936-4948.1988
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发表时间：
1988-11
影响因子：
5.3
通讯作者：
J. S. Robinson;D. Klionsky;L. Banta;S. Emr
中科院分区：
文献类型：
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作者：
J. S. Robinson;D. Klionsky;L. Banta;S. Emr
使用选择自发突变体，错误定位的液泡羧肽酶Y（CPY）-转化酶融合蛋白的细胞表面，我们确定了液泡蛋白靶向（VPT）突变体在25个新的VPT互补组。还获得了先前鉴定的八个vpt互补组（vpt 1至vpt 8）中的每一个中的其他等位基因。来自33个vpt互补组（vpt 1至vpt 33）的代表性等位基因显示出在几种天然液泡蛋白（包括可溶性水解酶CPY、蛋白酶A和蛋白酶B）的分选和加工中的缺陷。在33个互补组中，发现19个含有导致极端缺陷的突变等位基因。在这些突变体中，CPY以其高尔基复合体修饰的前体形式积累，由突变体细胞分泌。正常的蛋白质分泌似乎在vpt突变体中不受影响。从vpt突变体细胞中缺乏显著的胞质标记物泄漏表明与这些突变体相关的空泡蛋白分选缺陷不是由细胞溶解引起的。此外，vpt突变体分泌的是CPY、蛋白酶A、蛋白酶B的前体而不是成熟形式，这一观察结果与以下事实一致：错误定位发生在高尔基复合体特异性修饰后的某个阶段，但在这些酶的最终液泡分选之前。在大多数vpt突变体中，肺泡膜蛋白分选似乎不受影响。然而，一个子集的vpt突变体（vpt 11，vpt 16，vpt 18和vpt 33）被发现表现出缺陷的液泡膜标记酶，α-甘露糖苷酶的分选。在这些vpt突变体中，发现高达50%的α-甘露糖苷酶活性错误定位于细胞表面。七个vpt互补组（vpt 3，vpt 11，vpt 15，vpt 16，vpt 18，vpt 29和vpt 33）含有等位基因，导致条件致死表型;突变体对营养细胞生长的温度敏感。这种温度敏感的表型已被证明是隐性的，并在每种情况下与空泡蛋白分选缺陷共分离。四分体分析表明，vpt 3定位于染色体XV的右臂，vpt 15定位于染色体II的右臂。与其他突变体（vpl，sec，pep和end突变体）表现出液泡蛋白分选或功能缺陷的杂交揭示了这些不同基因组之间的一些重叠。总之，这些数据表明，超过50个基因产物直接或间接地参与了液泡蛋白分选的过程。
Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.