第三線嘌呤核苷二磷酸核醣化相似因子調控第一線嘌呤核苷二磷酸核醣化相似因子及其下游高爾基體蛋白Imh1在酵母菌中之功能探討

Abstract

高爾基體蛋白Imh1透過由鳥嘌呤核苷酸交換因子(GEF)Syt1所活化的第一線嘌呤核苷二磷酸核醣化相似因子 (Arl1)被吸引到反式高基氏體網路 (trans-Golgi network)上。由於實驗室先前發表的文章中提到Imh1可去抑制Ypt6缺發所造成的反向運輸缺陷，暗示著Imh1在反向運輸中的重要性。然而其詳細的功能目前訂不是很清楚。在結構上，Imh1可分為三個部分，包含胺基端、中間捲曲螺旋(coiled-coil)的結構以及羧基端。因此在本篇的研究中，我們希望去了解Imh1各個部分對於其功能的重要性 首先，我們發現到Imh1在內質網壓力下的SNARE蛋白的運輸中扮演重要的腳色，並進一步找到特別是其胺基端的磷酸化對於其功能的執行是重要的。此外，除了胺基端磷酸化很重要外，我們發現其中間的螺旋結構有助於去穩定Imh1的雙聚體的結構去幫助SNARE 蛋白的運輸。接著，我們利用一個會增強Imh1去形成二聚體能力的突變體Imh1-M4. 我們發現這個會增強Imh1形成二聚體能力的突變使得Imh1仍可在缺乏GEF或膜的彎曲程度有缺陷的情況下被吸引到反式高基氏體網路上。然而，這樣的突變並無法完全的彌補當Imh1與Arl1或膜上脂質的結合有問題時的Imh1功能。總結本篇，我們發現Imh1各個部分都擁有它特別的特性去協助Imh1整體的功能，包括胺基端被推測具有接取囊泡的功能以及中間的捲曲螺旋結構參與穩定其二聚體的形成，而其羧機端則是與Arl1和膜上脂質的交互作用有關。每個部分都有獨特的功能並需要彼此的合作來維持Imh1的功能。

Imh1 is a golgin protein recruited by active Arl1 through the induction of Syt1, an Arl1 guanine nucleotide exchange factor (GEF). It has been proposed to play a role in retrograde transport. Our previous study showed that Imh1 acts as a high-copy suppressor of retrograde transport defect in ypt6Δ. However, the precise function and regulation of Imh1 in yeast is less known. The structure of Imh1 is divided into three parts, including the N-terminal region, three coiled-coil domains and the C-terminal GRIP domain. In this study, we try to verify the contribution of each region on the function of Imh1. We first found that Imh1 is required for SNARE protein transport in response to Unfolded-Protein-Response (UPR). Moreover, this function is determined by the ER stress-driven phosphorylation of Imh1 N-terminus (Ser25 and Thr27). We also revealed that the Imh1 coiled-coil region is required for sustaining Imh1 dimerization and therefore supporting the SNARE transport. By using a forced dimerization mutant of Imh1 (Imh1-M4), we found that it can bypass the defect of dysfunctional GEF (sty1Δ cells) and the damage of sensing membrane curvature (drs2Δ cells). However, Imh1-M4 can only partially suppressed the defect in its Arl1 interaction and lipid-binding ability. Collectively, we demonstrate the importance of each region on Imh1 function, including N-terminus for vesicle capturing, coiled-coil region for stabilizing dimer structure and C-terminus for Arl1 interaction and lipid membrane association. All of these regions possess unique roles and need to orchestrate properly to support the Imh1 functions.