酵母菌第三腺核苷二磷酸核醣化因子相似蛋白之功能性探討

Abstract

腺核苷二磷酸醣化因子相似蛋白(ADP-ribosylation factor-like proteins, Arls)在反式高爾基體網絡(trans-Golgi network, TGN)的囊泡運輸中扮演著重要的角色，在酵母菌中，第三腺核苷二磷酸醣化因子相似蛋白(Arl3p)可以招集並活化第一腺核苷二磷酸醣化因子相似蛋白(Arl1p)，使得下游的三個蛋白Imh1p,Gas1p,Gga2p能夠執行其正常的功能。然而，目前對於Arl3p是怎麼樣去調控Arl1p或是Arl3p其獨自的功能並不很了解。本文利用酵母菌作為模式生物來探討Arl3p的功能，我們利用西式點墨法及螢光顯微鏡的觀察發現，當ARL3 與參與在核內體(endosome)與TGN間運輸的基因同時做基因剃除時會造成細胞質至液胞傳遞途徑(cytoplasm-to-vacuole targeting pathway, Cvt pathway)的缺陷產生。除此之外，我們也發現Arl1p下游蛋白的招集會在Arl3p與Arl1p同時在酸基端標記螢光蛋白時產生缺陷。但是這些在酸基端標記螢光蛋白Arl3p與Arl1p對於在細胞內的位置，或是其生物性功能都是正常的。最後，我們發現Arl3p會參與在Cvt pathway，並藉由未認定的方式去調控Arl1p。

Arf-like GTPases (Arl) play important roles in the vesicle trafficking at the trans-Golgi network (TGN). In yeast, Arl3p recruits Arl1p and the activated Arl1p in turn recruits a GRIP domain-containing protein Imh1p to regulate Golgi structure and function. Arl3p and Arl1p also participate in the transport of a GPI-anchored protein from the TGN to the plasma membrane to determine cell wall integrity. In addition, Arl1p also controls the membrane docking of Gga proteins to the TGN. However, the underlying mechanisms of how Arl3p regulate Arl1p or the unique function of Arl3p still remain unclear. In this study, we found that yeast cells with the double deletion of ARL3 and endosome-Golgi trafficking components lead to cytoplasm to vacuole targeting (Cvt) blockage. Notably, Arl1p would not show defect in Cvt pathway, suggesting that Arl3p participates in the Cvt pathway in an Arl1p-independent manner. In addition, we further demonstrated that the recruitment of Arl1p downstream effectors to the TGN was differentially affected when Arl3p and Arl1p were C-terminally fused with fluorescence protein. However, Arl3p and Arl1p fused with fluorescence protein at their C-terminus were, like un-tagged Arl3p and Arl1p, localized to the TGN and retained their biological activity for cell wall integrity. Together, our findings suggest that Arl3p may participate in the regulation of Cvt pathway, which is independent of Arl1p, and may control Arl1p activity by unidentified mechanism.