Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex.
Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex.
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DOI：
10.1091/mbc.e16-08-0570
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发表时间：
2016-12-15
影响因子：
3.3
通讯作者：
Emr SD
中科院分区：
文献类型：
--
作者：
Guiney EL;Klecker T;Emr SD
酿酒酵母中质膜蛋白的内吞作用需要它们被ART-Rsp 5泛素连接酶复合物泛素化。关于复合物如何与底物结合，人们知之甚少。Art 1的C-末端识别Mup 1通过三方ART分选信号：酸性补丁，在膜和底物赖氨酸附近。质膜（PM）蛋白的靶向内吞作用允许细胞调节其膜蛋白的补体以改变细胞外条件。对于多种PM蛋白，内吞作用的起始由泛素化触发。在酵母中，抑制蛋白相关的运输衔接子（ARTs）使一个单一的泛素连接酶，Rsp 5，特异性和选择性地靶向广泛的PM蛋白的泛素化和内吞作用。然而，允许ART特异性识别其适当底物的机制尚不清楚。我们提出的蛋氨酸通透酶Mup 1的Art 1-Rsp 5介导的泛素化和内吞作用所需的分子特征。遗传学、荧光显微镜和生物化学的组合揭示了Mup 1 N-末端胞质尾部中包含ART分选信号的三个关键特征：1）延伸的酸性斑块，2）靠近第一个Mup 1跨膜结构域，3）靠近泛素化赖氨酸。我们发现，功能相似的ART分选信号也需要第二个Art 1依赖性货物，Can 1的内吞作用，这表明一个共同的机制，识别Art 1基板。我们分离出两个单独的抑制突变的Art 1 C-末端结构域，等位基因特异性恢复内吞的两个Mup 1酸性补丁突变体，与Art 1 C-末端和Mup 1酸性补丁之间的相互作用一致。我们认为这种相互作用是Art 1-Rsp 5泛素化复合物募集所必需的。
Endocytosis of plasma membrane proteins in Saccharomyces cerevisiae requires their ubiquitination by the ART-Rsp5 ubiquitin ligase complex. Little is known about how the complex engages substrates. The Art1 C-terminus recognizes Mup1 via a tripartite ART sorting signal: an acidic patch, in proximity to the membrane and substrate lysines. Targeted endocytosis of plasma membrane (PM) proteins allows cells to adjust their complement of membrane proteins to changing extracellular conditions. For a wide variety of PM proteins, initiation of endocytosis is triggered by ubiquitination. In yeast, arrestin-related trafficking adaptors (ARTs) enable a single ubiquitin ligase, Rsp5, to specifically and selectively target a wide range of PM proteins for ubiquitination and endocytosis. However, the mechanisms that allow ARTs to specifically recognize their appropriate substrates are unknown. We present the molecular features in the methionine permease Mup1 that are required for Art1-Rsp5–mediated ubiquitination and endocytosis. A combination of genetics, fluorescence microscopy, and biochemistry reveals three critical features that comprise an ART sorting signal in the Mup1 N-terminal cytosolic tail: 1) an extended acidic patch, 2) in close proximity to the first Mup1 transmembrane domain, and 3) close to the ubiquitinated lysines. We show that a functionally similar ART sorting signal is also required for the endocytosis of a second Art1-dependent cargo, Can1, suggesting a common mechanism for recognition of Art1 substrates. We isolate two separate suppressor mutations in the Art1 C-terminal domain that allele-specifically restore endocytosis of two Mup1 acidic patch mutants, consistent with an interaction between the Art1 C-terminus and the Mup1 acidic patch. We propose that this interaction is required for recruitment of the Art1-Rsp5 ubiquitination complex.