Pal Bhupinder, Chan Nickie C, Helfenbaum Leon, Tan Kaeling, Tansey William P, Gething Mary-Jane
Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia.
Mol Biol Cell. 2007 Feb;18(2):426-40. doi: 10.1091/mbc.e06-04-0304. Epub 2006 Nov 15.
The Saccharomyces cerevisiae basic leucine zipper transcription factor Hac1p is synthesized in response to the accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), and it is responsible for up-regulation of approximately 5% of all yeast genes, including ER-resident chaperones and protein-folding catalysts. Hac1p is one of the most short-lived yeast proteins, having a half-life of approximately 1.5 min. Here, we have shown that Hac1p harbors a functional PEST degron and that degradation of Hac1p by the proteasome involves the E2 ubiquitin-conjugating enzyme Ubc3/Cdc34p and the SCF(Cdc4) E3 complex. Consistent with the known nuclear localization of Cdc4p, rapid degradation of Hac1p requires the presence of a functional nuclear localization sequence, which we demonstrated to involve basic residues in the sequence (29)RKRAKTK(35). Two-hybrid analysis demonstrated that the PEST-dependent interaction of Hac1p with Cdc4p requires Ser146 and Ser149. Turnover of Hac1p may be dependent on transcription because it is inhibited in cell mutants lacking Srb10 kinase, a component of the SRB/mediator module of the RNA polymerase II holoenzyme. Stabilization of Hac1p by point mutation or deletion, or as the consequence of defects in components of the degradation pathway, results in increased unfolded protein response element-dependent transcription and improved cell viability under ER stress conditions.
酿酒酵母碱性亮氨酸拉链转录因子Hac1p是在内质网(ER)腔中未折叠多肽积累时合成的,它负责上调约5%的酵母基因,包括内质网驻留伴侣蛋白和蛋白质折叠催化剂。Hac1p是酵母中寿命最短的蛋白质之一,半衰期约为1.5分钟。在这里,我们表明Hac1p含有一个功能性的PEST降解结构域,并且蛋白酶体对Hac1p的降解涉及E2泛素结合酶Ubc3/Cdc34p和SCF(Cdc4)E3复合物。与已知的Cdc4p核定位一致,Hac1p的快速降解需要功能性核定位序列的存在,我们证明该序列涉及序列(29)RKRAKTK(35)中的碱性残基。双杂交分析表明,Hac1p与Cdc4p的PEST依赖性相互作用需要Ser146和Ser149。Hac1p的周转可能依赖于转录,因为在缺乏Srb10激酶(RNA聚合酶II全酶的SRB/中介模块的一个组分)的细胞突变体中其周转受到抑制。通过点突变或缺失对Hac1p进行稳定化,或作为降解途径组分缺陷的结果,会导致未折叠蛋白反应元件依赖性转录增加,并在ER应激条件下提高细胞活力。