The C-terminal GGAP motif of Hsp70 mediates substrate recognition and stress response in yeast
The C-terminal GGAP motif of Hsp70 mediates substrate recognition and stress response in yeast
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Hsp70 的 C 端 GGAP 基序介导酵母中的底物识别和应激反应。
DOI：
10.1074/jbc.ra118.002691
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发表时间：
2018-11-16
影响因子：
4.8
通讯作者：
Perrett, Sarah
中科院分区：
文献类型：
--
作者：
Gong, Weibin;Hu, Wanhui;Perrett, Sarah
热休克蛋白70的高度保守的核苷酸和底物结合域的变构偶联已被深入研究。相反，Hsp 70的无序的、高度可变的C-末端区域的作用仍然不清楚。在许多真核Hsp 70中，末端C-末端EEVD基序与Hsp 70共分子伴侣的三肽重复结构域结合。在这里，我们发现，TVEEVD序列的酿酒酵母细胞质Hsp 70（Ssa 1）的功能作为一个SUMO相互作用的基序。在α-螺旋盖和末端C末端之间的第二个C-末端基序为15个氨基酸，先前在细菌和真核细胞器Hsp 70中鉴定，已知其通过与折叠客户瞬时相互作用来增强伴侣蛋白功能。利用结构分析、相互作用研究、原纤维形成测定和体内功能测定，我们研究了Ssa 1的α-螺旋束和C-末端无序区域在抑制朊病毒蛋白Ure 2原纤维形成中的各自贡献。我们的研究结果表明，尽管Ssa 1底物结合域（SBDα）的α-螺旋束不直接与Ure 2结合，但SBDα增强了Hsp 70抑制原纤维形成的能力。我们发现Ssa 1的20个残基的C-末端基序含有GGAP和GGAP样四肽重复序列，可以直接与Ure 2、Hsp 40共伴侣Ydj 1和α-突触核蛋白结合，但不与SUMO样蛋白SMT 3或BSA结合。缺失或取代的Ssa 1 GGAP基序损害酵母细胞耐受温度和细胞壁损伤应力。这项研究强调，热休克蛋白70的C-末端的GGAP基序是重要的底物识别和调解的热休克反应。
The allosteric coupling of the highly conserved nucleotide- and substrate-binding domains of Hsp70 has been studied intensively. In contrast, the role of the disordered, highly variable C-terminal region of Hsp70 remains unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds to the tetratricopeptide-repeat domains of Hsp70 co-chaperones. Here, we discovered that the TVEEVD sequence of Saccharomyces cerevisiae cytoplasmic Hsp70 (Ssa1) functions as a SUMO-interacting motif. A second C-terminal motif of ∼15 amino acids between the α-helical lid and the extreme C terminus, previously identified in bacterial and eukaryotic organellar Hsp70s, is known to enhance chaperone function by transiently interacting with folding clients. Using structural analysis, interaction studies, fibril formation assays, and in vivo functional assays, we investigated the individual contributions of the α-helical bundle and the C-terminal disordered region of Ssa1 in the inhibition of fibril formation of the prion protein Ure2. Our results revealed that although the α-helical bundle of the Ssa1 substrate-binding domain (SBDα) does not directly bind to Ure2, the SBDα enhances the ability of Hsp70 to inhibit fibril formation. We found that a 20-residue C-terminal motif in Ssa1, containing GGAP and GGAP-like tetrapeptide repeats, can directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and α-synuclein, but not to the SUMO-like protein SMT3 or BSA. Deletion or substitution of the Ssa1 GGAP motif impaired yeast cell tolerance to temperature and cell-wall damage stress. This study highlights that the C-terminal GGAP motif of Hsp70 is important for substrate recognition and mediation of the heat shock response.