Berkower C, Loayza D, Michaelis S
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Mol Biol Cell. 1994 Nov;5(11):1185-98. doi: 10.1091/mbc.5.11.1185.
STE6, a member of the ATP binding cassette (ABC) transporter superfamily, is a membrane protein required for the export of the a-factor mating pheromone in Saccharomyces cerevisiae. To initiate a study of the intracellular trafficking of STE6, we have examined its half-life and localization. We report here that STE6 is metabolically unstable in a wild-type strain, and that this instability is blocked in a pep4 mutant, suggesting that degradation of STE6 occurs in the vacuole and is dependent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissive temperature in mutants defective in delivery of proteins to the plasma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 appears to undergo constitutive internalization from the plasma membrane, as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (half- and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is found in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pattern is consistent with the view that most of the STE6 molecules present in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vacuole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indicating that it can accumulate in the plasma membrane when internalization is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transient residence at the plasma membrane; an alternative model, in which STE6 circumvents the plasma membrane, is also discussed.
STE6是ATP结合盒（ABC）转运蛋白超家族的成员之一，是酿酒酵母中α因子交配信息素输出所必需的膜蛋白。为了启动对STE6细胞内运输的研究，我们检测了它的半衰期和定位。我们在此报告，STE6在野生型菌株中代谢不稳定，而在pep4突变体中这种不稳定性被阻断，这表明STE6的降解发生在液泡中，并且依赖于液泡蛋白酶。与STE6通过从质膜的内吞作用被转运至液泡的模型一致，我们发现，在蛋白质向质膜转运缺陷（sec6）或内吞作用缺陷（end3和end4）的突变体中，在非允许温度下STE6的降解显著减少。虽然STE6似乎像信息素受体STE2和STE3一样从质膜进行组成型内化，但我们发现另外两种蛋白质，即质膜ATP酶（PMA1）和通用氨基酸通透酶（GAP1），比STE6稳定得多，这表明在液泡中的快速周转并非酵母中所有质膜蛋白的共同命运。对STE6部分分子（半分子和四分之一分子）的研究表明，STE6的两个半部分都包含足以介导内化的信息。通过间接免疫荧光检测STE6的定位表明，STE6以点状、可能是囊泡状的细胞内模式存在，这与PMA1特有的边缘染色模式不同。这种点状模式与以下观点一致，即在任何给定时刻细胞中存在的大多数STE6分子可能正在往返于质膜的途中。在pep4突变体中，STE6集中在液泡中，这进一步证明液泡是STE6降解的部位，而在end4突变体中，STE6表现出边缘染色，表明当内化被阻断时它可以在质膜中积累。综上所述，本文给出的结果表明，STE6首先转运至质膜，随后经历内吞作用并在液泡中降解，可能仅在质膜处短暂停留；还讨论了另一种模型，即STE6绕过质膜。
