Molecular mechanisms of protein glycosylation and trafficking

项目摘要 这项建议的目的是获得对蛋白质糖基化的机制和病理学的了解。 和贩卖人口。在前一个供资周期中，我们为这一进程作出了重要贡献。我们 确定了跨膜结构域插入酶EMC复合体的结构，揭示了一种拉长的 结构的跨膜区中的空腔，可以容纳弱疏水 跨膜螺旋。我们已经解决了蛋白质N-糖基转移酶(OST)的结构和 蛋白O-甘露糖基转移酶Pmt1-Pmt2，揭示其催化的进化保守的GT-C折叠 亚单位。蛋白质的运输需要形成脂泡，这一过程依赖于脂膜转运酶的活性。 以建立双层的两个小叶之间的成分不对称。在这方面，我们有 测定了三类酵母脂转移酶的结构。这项续签提案延续了我们的 最主要的目标是了解蛋白质的糖基化和运输。我们建议解决两个具体问题 知识差距：两种蛋白甘露糖转移酶的结构和机制，以及最近 发现三元蛋白复合体ARL1-Gea2-Drs2将膜曲率与脂质翻转活性偶联 形成，从而促进下游的囊泡发芽过程，以进行蛋白质和膜的运输。 Drs2是一种磷脂酰丝氨酸翻转酶，是形成AP-1/cathrin包被的囊泡所必需的。 在跨高尔基体网络(TGN)和早期内小体之间来回。小的GTP酶ARL1是一种 ADP-核糖化因子(Arf)家族的成员，由Arf鸟嘌呤核苷酸交换激活 因数Gea2。ARL1只在TGN运行，是ARF家族中最不为人所知的成员。 我们将在体外重组三元配合物，并进行全面的结构-功能研究。蛋白 糖基化和转运与肿瘤的发生和癌症进展密切相关。我们的机械师 研究将填补这些基本的知识空白，我们的派生结构可能会促进发展 用于癌症治疗的小分子。

Project Summary The objective of this proposal is to gain mechanistic and pathological understanding of protein glycosylation and trafficking. During the previous funding cycle, we have made important contributions to this process. We have determined the structure of the transmembrane domain insertase EMC complex, revealing an elongated cavity in the transmembrane region of the structure that can accommodate a weakly hydrophobic transmembrane helix. We have solved the structures of the protein N-glycosyltransferase (OST) and the protein O-mannosyltransferase Pmt1-Pmt2, revealing the evolutionarily conserved GT-C folds of their catalytic subunits. Protein trafficking requires lipid vesicle formation, a process that is dependent on lipid flippase activity to establish compositional asymmetry between the two leaflets of the bilayer. In this regard, we have determined the structures of all three classes of yeast lipid flippases. This renewal proposal continues our overarching goal to understand protein glycosylation and trafficking. We propose to address two specific knowledge gaps: the structure and mechanism of two protein mannosyltransferases, and how the recently discovered ternary protein complex Arl1-Gea2-Drs2 couples lipid flipping activity with membrane curvature formation, thereby facilitating the downstream vesicle budding process for protein and membrane trafficking. Drs2 is a phosphatidylserine flippase required for the formation of AP-1/clathrin-coated vesicles that travel back and forth between the trans-Golgi network (TGN) and early endosomes. The small GTPase Arl1 is a member of the ADP-ribosylation factor (Arf) family that is activated by the Arf guanine nucleotide exchange factor Gea2. Arl1 operates exclusively in the TGN and is the least well-understood member of the Arf family. We will reconstitute the ternary complex in vitro and perform a comprehensive structure-function study. Protein glycosylation and trafficking is intimately linked to tumorigenesis and cancer progression. Our mechanistic studies will fill these fundamental knowledge gaps, and our derived structures may facilitate the development of small molecules for cancer treatment.