Robinson J S, Klionsky D J, Banta L M, Emr S D
Division of Biology, California Institute of Technology, Pasadena 91125.
Mol Cell Biol. 1988 Nov;8(11):4936-48. doi: 10.1128/mcb.8.11.4936-4948.1988.
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.
通过选择使液泡羧肽酶Y（CPY）-转化酶融合蛋白错误定位到细胞表面的自发突变体，我们在25个新的液泡蛋白靶向（vpt）互补组中鉴定出了vpt突变体。还获得了先前鉴定的8个vpt互补组（vpt1至vpt8）中每个组的额外等位基因。来自33个vpt互补组（vpt1至vpt33）中每个组的代表性等位基因显示在几种天然液泡蛋白的分选和加工过程中存在缺陷，包括可溶性水解酶CPY、蛋白酶A和蛋白酶B。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以其经高尔基体复合体修饰的前体形式积累，并由突变细胞分泌。vpt突变体中正常的蛋白质分泌似乎未受影响。vpt突变体细胞中胞质标记物没有明显泄漏，这表明与这些突变体相关的液泡蛋白分选缺陷不是由细胞裂解导致的。此外，观察到CPY、蛋白酶A、蛋白酶B的前体而非成熟形式从vpt突变体中分泌，这与错误定位发生在高尔基体复合体特异性修饰之后，但在这些酶最终液泡分选之前这一事实是一致的。大多数vpt突变体中液泡膜蛋白分选似乎未受影响。然而，发现一部分vpt突变体（vpt11、vpt16、vpt18和vpt33）在液泡膜标记酶α-甘露糖苷酶的分选方面存在缺陷。在这些vpt突变体中，高达50%的α-甘露糖苷酶活性被发现错误定位到细胞表面。7个vpt互补组（vpt3、vpt11、vpt15、vpt16、vpt18、vpt29和vpt33）含有导致条件致死表型的等位基因；这些突变体在营养细胞生长方面对温度敏感。这种温度敏感表型已被证明是隐性的，并且在每种情况下都与液泡蛋白分选缺陷共分离。四分体分析表明，vpt3定位于第十五号染色体的右臂，vpt15定位于第二号染色体的右臂。与其他在液泡蛋白分选或功能方面表现出缺陷的突变体（vpl、sec、pep和end突变体）进行杂交，揭示了这些不同基因集之间的一些重叠。总之，这些数据表明，超过50种基因产物直接或间接参与了液泡蛋白分选过程。
