Wilsbach K, Payne G S
Molecular Biology Institute, University of California, Los Angeles 90024.
EMBO J. 1993 Aug;12(8):3049-59. doi: 10.1002/j.1460-2075.1993.tb05974.x.
The KEX2-encoded endoprotease of Saccharomyces cerevisiae resides in the Golgi complex where it participates in the maturation of alpha-factor mating pheromone precursor. Clathrin heavy chain gene disruptions cause mislocalization of Kex2p to the cell surface and reduce maturation of the alpha-factor precursor. Based on these findings, a genetic screen has been devised to isolate mutations that affect retention of Kex2p in the Golgi complex. Two alleles of a single genetic locus, lam1 (lowered alpha-factor maturation), have been isolated, which result in inefficient maturation of alpha-factor precursor. In lam1 cells, Kex2p is not mislocalized to the cell surface but is abnormally unstable. Normal stability is restored by the pep4 mutation which reduces the activity of vacuolar proteases. In contrast, the pheromone maturation defect is not corrected by pep4. Organelle fractionation by sucrose density gradient centrifugation shows that Kex2p is not retained in the Golgi complex of lam1 cells. Vacuolar protein precursors are secreted by lam1 mutants, revealing another sorting defect in the Golgi complex. Genetic complementation reveals that lam1 is allelic to the VPS1 gene, which encodes a dynamin-related GTPase. These results indicate that Vps1p is necessary for membrane protein retention in a late Golgi compartment.
酿酒酵母中由KEX2编码的内切蛋白酶定位于高尔基体复合体，在那里它参与α-因子交配信息素前体的成熟过程。网格蛋白重链基因的破坏会导致Kex2p错误定位于细胞表面，并减少α-因子前体的成熟。基于这些发现，设计了一种遗传筛选方法来分离影响Kex2p在高尔基体复合体中保留的突变。已分离出一个单一基因座的两个等位基因lam1（α-因子成熟降低），它们导致α-因子前体的成熟效率低下。在lam1细胞中，Kex2p没有错误定位于细胞表面，但异常不稳定。pep4突变可恢复正常稳定性，该突变会降低液泡蛋白酶的活性。相反，信息素成熟缺陷不能通过pep4得到纠正。通过蔗糖密度梯度离心进行细胞器分级分离表明，Kex2p没有保留在lam1细胞的高尔基体复合体中。lam1突变体分泌液泡蛋白前体，这揭示了高尔基体复合体中的另一种分选缺陷。遗传互补表明，lam1与VPS1基因等位，该基因编码一种与发动蛋白相关的GTP酶。这些结果表明，Vps1p对于膜蛋白保留在高尔基体晚期区室中是必需的。
