探討磷酸化對於GTP酶活化蛋白Gcs1之影響

Abstract

腺嘌呤核苷二磷酸核糖化因子(Arfs)與腺嘌呤核苷二磷酸核糖化相似因子(Arls)被認為在囊泡運輸中扮演著重要的角色。在釀酒酵母菌中，第一腺嘌呤核苷二磷酸核糖化相似因子(Arl1)的鳥嘌呤核苷酸結合循環是由鳥嘌呤核苷酸交換因子(GEF) Syt1以及GTP酶活化蛋白(GAP) Gcs1進行轉換。然而，Gcs1接觸高基氏體並促進Arl1的GTP水解是如何受到調控的詳細機制仍然不清楚。在這裡，我們發現低水平的葡萄糖會誘發Gcs1的磷酸化。被質譜儀辨認到的那些磷酸化位點能調降Gcs1對於Arl1的GAP活性。再者，我們注意到Snf1與Rim15兩者所調節的路徑，在葡萄糖剝奪的環境中，都有助於Gcs1的磷酸化。此外，我們也演示了Gcs1在衣黴素處理後所導致的內質網壓力下會被磷酸化。藉由質譜儀，我們在Gcs1上鑑定到幾個對內質網壓力反應的新磷酸化位點，包含S157，T161，S321以及S322。我們觀察到被磷酸化的Gcs1能幫助Arl1與Imh1在未折疊蛋白質反應下進一步的募集到高基氏體。有趣的是，我們發現Gcs1在內質網壓力下磷酸化的引信並不依賴Ire1-Hac1這條為人熟知的訊息傳遞路徑，反而是經由Slt2相關的信號。並且，剔除Slt2減緩了在未折疊蛋白質反應下Imh1募集量的增加。因此，我們推測Gcs1磷酸化在空間與時間上的調節可能控制了Arl1在高基氏體上的活化狀態。

ADP-ribosylation factors (Arfs) and Arf-like proteins (Arls) are known to play critical roles in vesicle trafficking. In Saccharomyces cerevisiae, the guanine nucleotide-binding cycle of Arl1 is converted by the guanine nucleotide exchange factor (GEF), Syt1, and the GTPase activating protein (GAP), Gcs1. However, the detailed mechanism of how Gcs1 is regulated to access the Golgi membrane and promote GTP-hydrolysis of Arl1 remains unclear. Here, we find that the low level of glucose triggers Gcs1 phosphorylation. These phosphorylation sites recognized by mass spectrometry down-regulate the GAP activity of Gcs1 toward Arl1. Moreover, we notice that both the Snf1 and the Rim15 mediated pathways contribute to Gcs1 phosphorylation under glucose deprivation. Besides, we also demonstrate that Gcs1 is phosphorylated upon tunicamycin treatment, which causes ER stress. By mass spectrometry, we identify several new phosphorylation sites on Gcs1 in response to ER stress, including S157, T161, S321, and S322. We observe that the phosphorylated Gcs1 supports the further recruitment of Arl1 and Imh1 to Golgi upon UPR. Interestingly, we find that the trigger of Gcs1 phosphorylation upon ER stress was not dependent on a well-known Ire1-Hac1 signaling pathway, but rather Slt2 related signaling. Furthermore, Slt2 deletion attenuates the increase of Imh1 recruitment upon UPR. Thus, we infer that the spatial and temporal regulation of Gcs1 phosphorylation may modulate the activation status of Arl1 at the Golgi.