Darsow T, Burd C G, Emr S D
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668, USA.
J Cell Biol. 1998 Aug 24;142(4):913-22. doi: 10.1083/jcb.142.4.913.
The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Delta cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein-protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7-amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor-dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.
在出芽酵母酿酒酵母中，新合成蛋白质通过液泡蛋白分选途径进行运输需要两种不同的靶标可溶性NSF附着蛋白受体（t-SNARE）蛋白，即Pep12p和Vam3p。Pep12p定位于液泡前内体，其活性是蛋白质通过明确途径（羧肽酶Y（CPY）途径）从高尔基体运输到液泡所必需的。Vam3p定位于液泡，在那里它介导来自CPY途径和最近描述的碱性磷酸酶（ALP）途径的货物运输。令人惊讶的是，尽管它们在液泡蛋白分选方面具有细胞器特异性功能，但VAM3的过表达可以抑制pep12Δ细胞的蛋白质分选缺陷。基于这一观察结果，我们开发了一种遗传筛选方法，以鉴定Vam3p中的结构域（例如，定位和/或特定的蛋白质-蛋白质相互作用结构域），使其能够有效地替代Pep12p。使用这种筛选方法，我们在Vam3p中鉴定出一个7个氨基酸序列的突变，该突变导致Vam3p从ALP途径错分到CPY途径，在那里它可以在液泡前内体替代Pep12p。该区域包含一个酸性双亮氨酸序列，与酵母和哺乳动物系统中AP-3衔接蛋白依赖性运输所需的分选信号密切相关。此外，AP-3功能的破坏也导致野生型Vam3p能够补偿pep12突变体，这表明AP-3通过双亮氨酸信号介导Vam3p的分选。总之，这些数据首次鉴定了t-SNARE蛋白中衔接蛋白特异性分选信号，并表明AP-3依赖性的Vam3p分选作用是限制其与特定细胞器辅助蛋白的相互作用，从而调节其功能。诸如Vam3p之类的货物通过AP-3依赖性途径的调节运输可能在维持液泡独特的组成、功能和形态方面发挥重要作用。
