Identification of discrete sites in Yip1A necessary for regulation of endoplasmic reticulum structure.
Identification of discrete sites in Yip1A necessary for regulation of endoplasmic reticulum structure.
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DOI：
10.1371/journal.pone.0054413
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Lee TH
中科院分区：
文献类型：
--
作者：
Dykstra KM;Ulengin I;Delrose N;Lee TH
特化细胞的内质网（ER）在结构组织上可以发生戏剧性的变化，包括形成同心螺旋。我们以前报道过，Yip 1A，酵母和哺乳动物之间保守的一个完整的膜蛋白，耗尽引起ER螺旋的形成，让人想起在专门的细胞。Yip 1A及其在ER和早期高尔基体之间的酵母同源物Yip 1 p循环涉及许多不同的运输步骤，并与包括Yif 1 p/Yif 1A和Ypt 1/Ypt 31 Rab GTP酶在内的一组保守的结合伴侣相互作用。在这里，我们进行了Yip 1A的突变分析，以了解它如何调节ER螺旋形成。大多数Yip 1A的胞质结构域是突变的，而跨膜（TM）结构域，特别是预测的TM螺旋3和4内的残基，对诱变敏感。综合分析表明，两个离散的功能所需的决定因素。一个是E95和侧翼残基L92和L96内的胞质结构域;另一个是K146和附近的残基V152内的TM结构域。值得注意的是，确定的决定因素密切对应于两个位点，以前发现是必不可少的酵母活力（E76和K130在Yip 1 p对应E95和K146在Yip 1A，分别）。相反，第三个位点（E89）也是酵母活力（E70在Yip 1 p）所必需的，它被用来调节螺旋形成。早期的研究表明，E76（E95）是结合Yif 1 p或Ypt 1 p/Ypt 31 p所必需的，而E70（E89）是必需的。总的来说，这些研究结果表明，Yip 1A结合其已建立的结合伙伴的能力可能是从其控制ER螺旋形成的能力中分离出来的。作为支持，YiflA敲除不引起ER螺旋形成。因此，Yip 1A可以使用本文鉴定的位点与新的结合伴侣相互作用以调节ER膜组织。
The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.
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