The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis
The internalization of yeast Ste6p follows an ordered series of events involving phosphorylation, ubiquitination, recognition and endocytosis
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DOI：
10.1111/j.1600-0854.2004.00168.x
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发表时间：
2004-03-01
期刊：
影响因子：
4.5
通讯作者：
Michaelis, S
中科院分区：
文献类型：
--
作者：
Kelm, KB;Huyer, G;Michaelis, S
细胞膜蛋白内化的一般途径包括磷酸化、泛素化、识别和内吞作用，最近在酵母的多项研究中出现。我们将这一系列事件称为PURE途径。在这里，我们研究了酵母a因子转运蛋白Ste6p（一种atp结合盒蛋白）是否利用了PURE途径。先前有研究表明，Ste6p连接区域中52个氨基酸序列的缺失可以阻断泛素化和内吞作用（Kolling R, Losko S. EMBO J 1997; 16:2251-2261）。利用野生型和突变型gfp标记的Ste6p，我们在A框中发现了两个残基（T-613和S-623），它们可能是Ste6p磷酸化的位点，对内化很重要。这些残基突变为丙氨酸阻断了Ste6p的泛素化和内吞作用，类似于删除整个A盒的效果，而用谷氨酸替代（模拟磷酸化）抑制了泛素化和内吞缺陷。重要的是，单泛素与Ste6p- t (613) a、S(623) a或Ste6p- deltaa的c端翻译融合恢复了内吞作用，这提供了强有力的证据，证明磷酸化的作用是指导泛素化，而泛素化反过来又是Ste6p内化的关键信号。我们还在连接体中发现了多个（五个）赖氨酸残基，这些残基对Ste6p泛素化很重要。我们的研究结果表明，Ste6p遵循PURE途径，并且gfp标记的Ste6p为酵母内吞作用和内吞后事件的研究提供了一个强大的模型蛋白。
A general pathway for the internalization of plasma membrane proteins that involves phosphorylation, ubiquitination, recognition and endocytosis has recently emerged from multiple studies in yeast. We refer to this series of events as the PURE pathway. Here we investigate whether the yeast a-factor transporter Ste6p, an ATP-binding cassette protein, utilizes the PURE pathway. Deletion of a 52-amino acid sequence (the 'A box') within the linker region of Ste6p has previously been shown to block ubiquitination and endocytosis (Kolling R, Losko S. EMBO J 1997; 16:2251-2261). Using wild-type and mutant forms of GFP-tagged Ste6p, we identified two residues (T-613 and S-623) within the A box as likely sites of Ste6p phosphorylation important for internalization. Mutation of these residues to alanine blocked ubiquitination and endocytosis of Ste6p, similar to the effect of deleting the entire A box, while substitution with glutamic acid (to mimic phosphorylation) suppressed the ubiquitination and endocytic defects. Importantly, a translational fusion of monoubiquitin to the C-terminus of Ste6p-T(613)A, S(623)A or Ste6p-DeltaA restored endocytosis, providing strong evidence that the role of phosphorylation is to direct ubiquitination, which in turn is a critical signal for Ste6p internalization. We also identified multiple (five) lysine residues in the linker that are important for Ste6p ubiquitination. Our results demonstrate that Ste6p follows the PURE pathway and that GFP-tagged Ste6p provides a powerful model protein for studies of endocytosis and post-endocytic events in yeast.