探討在壓力情況下磷酸化高基體體蛋白Imh1及腺嘌呤核苷二磷酸核醣化因子GTPase活化蛋白Gcs1對高基體運輸的調控

Abstract

小GTPase坐落在高基氏體上，是作為調控細胞內蛋白質運輸的重要蛋白。小GTPase的活性受到鳥嘌呤核苷酸交換因子(Guanine nucleotide exchange factors, GEFs)以及GTPase活化蛋白(GTPase activating protein, GAP)的調控。在發酵酵母(Saccharomyces Cerevisiae)中，腺嘌呤核苷二磷酸核醣化因子相似蛋白1 (Arl1)是個在細胞壓力下，對於囊泡的逆向運輸扮演重要角色的小GTPase。目前已知Arl1的活性受到GEF蛋白Syt1以及GAP蛋白Gcs1的調控。活化的Arl1會招募高基氏體蛋白(Golgin) Imh1到高基氏體、調控糖基磷脂酰肌醇锚定蛋白(GPI-anchor protein) Gas1的運輸以及幫助衔接蛋白(adaptor protein) Gga2的運輸。在過去的研究中，我們發現在內質網壓力中，Arl1會被高度活化並招募更多的Imh1到高基氏體，同時，Imh1會被磷酸化並共同調控SNARE蛋白Snc1的逆向運輸。而在本篇研究中，我們更進一步發現，在缺乏糖分的情況下，Imh1會被磷酸化並促使其離開高基氏體，而這樣的磷酸化調控參與在細胞維持高基氏體恆定的機制中。而回到內質網壓力中，我們過去已知是磷酸化誘導的Syt1 (Arl1的GEF蛋白)活化來促使高度活化的Arl1。然而，對於Gcs1在內質網壓力中的角色目前並不清楚。在本篇研究中，我們發現透過磷酸化，Gcs1也參與在SNARE蛋白Scn1的運輸中。然而磷酸化的Gcs1並不影響其自身的酵素活性，代表其非透過改變Arl1的活性來影響Snc1運輸。我們發現磷酸化會影響Gcs1與Snc1之間的蛋白交互作用。綜合以上，我們透過這些研究了解到高基氏體蛋白Imh1以及GTPase活化蛋白Gcs1在細胞壓力下的調控以及所扮演的角色。

Small GTPases are responsible for protein transport in the cells. They localize on the Golgi, which serves as a protein sorting hub. The activity of small GTPases is regulated by Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Here, we focus on the small GTPase, Arl1, which localizes at the late-Golgi and is responsible for the protein retrograde transport under cellular stresses. Arl1 is activated by the known GEF, Syt1, and Gcs1 acts as a GAP for Arl1 to facilitate its inactivation. The downstream effectors of Arl1 include Golgin Imh1, GPI-anchor protein Gas1, and adaptor protein Gga2. In our previous finding, the activated Arl1 recruits Imh1 to the Golgi, and the Golgi-localized Imh1 then tethers the SNARE proteins to maintain retrograde transport under ER stress conditions. In addition to the ER stress, we further found that Imh1 also plays an important role under glucose deprivation. In this study, we revealed that the localization of Imh1 is regulated by the phosphorylation status under glucose deprivation, and the localization change is related to the maintenance of Golgi compartment under glucose deprivation. These results emphasize the role of Arl1 and Imh1 under stress conditions. Besides, we further investigate the role of Gcs1, the GAP of Arl1, under ER stress conditions. We previously reported that the phosphorylated GEF Syt1 is required for enhancing the vesicle transport function of Arl1 and Imh1, but the role of Gcs1 under ER stress is still unclear. In this study, we found that Gcs1 is phosphorylated under ER stress, and the phosphorylated Gcs1 defects its function in SNARE protein transport. Although Gcs1 is the GAP of Arl1, we surprisingly found that the phosphorylated Gcs1-regulated SNARE transport is independent of Arl1 activity but dependent on the direct interaction with Snc1. In summary, we reveal the function and regulation of Golgin Imh1 and Arf-GAP Gcs1 under different cellular stress.