Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2013;8(1):e54413. doi: 10.1371/journal.pone.0054413. Epub 2013 Jan 14.
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.
内质网（ER）的特化细胞可以经历结构组织的显著变化，包括形成同心的漩涡。我们之前报道过，耗尽整膜蛋白 Yip1A，酵母和哺乳动物之间保守的一种，会导致 ER 漩涡形成，类似于特化细胞中观察到的。Yip1A 和它的酵母同源物 Yip1p 在 ER 和早期高尔基体之间循环，已经涉及到许多不同的运输步骤，并与一组保守的结合伙伴相互作用，包括 Yif1p/Yif1A 和 Ypt1/Ypt31 Rab GTPases。在这里，我们对 Yip1A 进行了突变分析，以深入了解它如何调节 ER 漩涡的形成。Yip1A 的大部分细胞质结构域是可有可无的，而跨膜（TM）结构域，特别是预测的 TM 螺旋 3 和 4 内的残基，对突变很敏感。全面分析揭示了两个离散的功能必需决定因素。一个是位于细胞质结构域内的 E95 和侧翼残基 L92 和 L96；另一个是位于 TM 结构域内的 K146 和附近的残基 V152。值得注意的是，鉴定出的决定因素与先前发现的对酵母生存至关重要的两个位点（Yip1p 中的 E76 和 K130 分别对应于 Yip1A 中的 E95 和 K146）非常接近。相比之下，第三个对酵母生存至关重要的位点（E89）（Yip1p 中的 E70）对于漩涡形成的调节是可有可无的。早期的工作表明，E76（E95）对于结合 Yif1p 或 Ypt1p/Ypt31p 是可有可无的，而 E70（E89）是必需的。总的来说，这些发现表明，Yip1A 结合其已建立的结合伙伴的能力可能与其控制 ER 漩涡形成的能力脱钩。支持这一观点的是，Yif1A 的敲低并没有导致 ER 漩涡的形成。因此，Yip1A 可能使用此处确定的位点与一个新的结合伙伴相互作用，以调节 ER 膜的组织。
