Role for the ubiquitin-proteasome system in the vacuolar degradation of Ste6p, the a-factor transporter in Saccharomyces cerevisiae.
Role for the ubiquitin-proteasome system in the vacuolar degradation of Ste6p, the a-factor transporter in Saccharomyces cerevisiae.
复制标题
泛素蛋白酶体系统在酿酒酵母 a 因子转运蛋白 Ste6p 液泡降解中的作用。
DOI：
10.1128/mcb.18.2.779
复制
发表时间：
1998
影响因子：
5.3
通讯作者：
Michaelis,S
中科院分区：
文献类型：
--
作者：
Loayza,D;Michaelis,S
Ste6p 是酿酒酵母中的 a 因子转运蛋白，是一种多跨膜蛋白，具有 12 个跨膜跨度和两个胞质 ATP 结合域。 Ste6p 属于 ATP 结合盒 (ABC) 超家族，为检查酵母中复杂多面体膜蛋白的细胞内运输提供了一个极好的模型。先前的研究表明，Ste6p 从质膜经历组成型内吞作用，然后输送到液泡，在液泡中以 Pep4p 依赖性方式降解，尽管只有一小部分 Ste6p 暴露于 Pep4p 依赖性蛋白酶所在的液泡腔。已知 Ste6p 被泛素化，这种修饰可能促进其内吞作用。在本研究中，我们进一步研究了Ste6p的细胞内运输，重点关注泛素蛋白酶体机制在Ste6p代谢降解中的作用。我们通过脉冲追踪分析证明，在蛋白酶体活性缺陷的突变体（doa4andpre1,2）中，Ste6p 的降解受到损害。同样，通过免疫荧光，我们观察到 Ste6p 在 thedoa4 突变体的液泡中积累，就像在液泡蛋白酶缺陷的 pep4 突变体中一样。与我们的结果一致的一个模型是，Ste6p 的降解（其中大部分暴露于细胞质）需要细胞质蛋白酶体降解机制和液泡腔蛋白酶的活性，以协同方式发挥作用。或者，我们讨论了第二种模型，泛素蛋白酶体系统可能间接影响 Ste6p 的 Pep4p 依赖性液泡降解。这项研究表明，Ste6p 的独特之处在于两个独立的降解系统（液泡 Pep4p 依赖性蛋白酶和胞质蛋白酶体）都直接或间接参与单一底物的代谢降解。
Ste6p, the a-factor transporter inSaccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4andpre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in thedoa4mutant, as it does in the vacuolar protease-deficientpep4mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.