Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo.
Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo.
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剖析体内共陪伴 Hsp70 网格蛋白脱壳活性的 Swa2p/auxilin 结构域要求。
DOI：
10.1091/mbc.e06-02-0106
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发表时间：
2006
影响因子：
3.3
通讯作者：
Graham,ToddR
中科院分区：
文献类型：
--
作者：
Xiao,Jing;Kim,LeslieS;Graham,ToddR
生长素家族的J结构域蛋白将Hsp 70装载到网格蛋白包被的囊泡（CCV）上以驱动去包被。在体外，生长素的功能需要其能够结合网格蛋白并通过其J结构域刺激Hsp 70 ATP酶活性。为了在体内测试这些要求，我们对Swa 2 p（酵母生长素直系同源物）进行了突变分析。Swa 2 p是一种具有三个N-末端网格蛋白结合（CB）基序、一个泛素结合（乌巴）结构域、一个三肽重复（TPR）结构域和一个C-末端J-结构域的模块蛋白。在体外，网格蛋白结合是由多种弱相互作用介导的，但缺少两个CB基序和乌巴结构域的Swa 2 p截短在体内保留了几乎全部功能。删除所有CB基序强烈废除网格蛋白解体，但不消除体内Swa 2 p功能。令人惊讶的是，突变的J-结构域内的不变的HPD基序AAA仅部分影响Swa 2 p的功能。类似地，TPR点突变（G388 R）引起适度表型。然而，当这些TPR和J突变组合时，Swa 2 p功能被消除。TPR和J结构域在功能上不是冗余的，因为缺失任一结构域都会使Swa 2 p失去功能。这些数据表明，TPR和J-结构域合作的二分相互作用与热休克蛋白70调节其活性的网格蛋白解体。
The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.