Stepp J D, Huang K, Lemmon S K
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
J Cell Biol. 1997 Dec 29;139(7):1761-74. doi: 10.1083/jcb.139.7.1761.
A novel clathrin adaptor-like complex, adaptor protein (AP)-3, has recently been described in yeast and in animals. To gain insight into the role of yeast AP-3, a genetic strategy was devised to isolate gene products that are required in the absence of the AP-3 mu chain encoded by APM3. One gene identified by this synthetic lethal screen was VPS45. The Vps pathway defines the route that several proteins, including carboxypeptidase Y, take from the late Golgi to the vacuole. However, vacuolar alkaline phosphatase (ALP) is transported via an alternate, intracellular route. This suggested that the apm3-Delta vps45 synthetic phenotype could be caused by a block in both the alternate and the Vps pathways. Here we demonstrate that loss of function of the AP-3 complex results in slowed processing and missorting of ALP. ALP is no longer localized to the vacuole membrane by immunofluorescence, but is found in small punctate structures throughout the cell. This pattern is distinct from the Golgi marker Kex2p, which is unaffected in AP-3 mutants. We also show that in the apm3-Delta mutant some ALP is delivered to the vacuole by diversion into the Vps pathway. Class E vps mutants accumulate an exaggerated prevacuolar compartment containing membrane proteins on their way to the vacuole or destined for recycling to the Golgi. Surprisingly, in AP-3 class E vps double mutants these proteins reappear on the vacuole. We suggest that some AP-3-dependent cargo proteins that regulate late steps in Golgi to vacuole transport are diverted into the Vps pathway allowing completion of transfer to the vacuole in the class E vps mutant.
最近在酵母和动物中发现了一种新型网格蛋白衔接蛋白样复合物，即衔接蛋白（AP）-3。为深入了解酵母AP-3的作用，设计了一种遗传策略来分离在缺乏由APM3编码的AP-3μ链时所需的基因产物。通过这种合成致死筛选鉴定出的一个基因是VPS45。Vps途径定义了几种蛋白质（包括羧肽酶Y）从晚期高尔基体到液泡的运输路线。然而，液泡碱性磷酸酶（ALP）通过另一条细胞内途径运输。这表明apm3Δvps45合成表型可能是由替代途径和Vps途径的阻断引起的。在这里，我们证明AP-3复合物功能丧失导致ALP加工减慢和分选错误。通过免疫荧光法，ALP不再定位于液泡膜，而是在整个细胞中的小斑点结构中发现。这种模式与高尔基体标志物Kex2p不同，Kex2p在AP-3突变体中不受影响。我们还表明，在apm3Δ突变体中，一些ALP通过转入Vps途径被递送至液泡。E类vps突变体在其液泡或注定循环回高尔基体的途中积累了一个含有膜蛋白的过度扩大的前液泡区室。令人惊讶的是，在AP-3 E类vps双突变体中，这些蛋白质重新出现在液泡上。我们认为，一些调节高尔基体到液泡运输后期步骤的AP-3依赖性货物蛋白被转入Vps途径，从而允许在E类vps突变体中完成向液泡的转运。
