Molecular mechanisms of protein glycosylation and trafficking

项目概要 该提案的目的是获得对蛋白质糖基化的机制和病理学理解 和贩运。在上一个融资周期中，我们为这一进程做出了重要贡献。我们 确定了跨膜结构域插入酶 EMC 复合物的结构，揭示了一个细长的 结构跨膜区域中的空腔，可以容纳弱疏水性 跨膜螺旋。我们已经解析了蛋白质N-糖基转移酶（OST）的结构和 蛋白质 O-甘露糖基转移酶 Pmt1-Pmt2，揭示了其催化的进化上保守的 GT-C 折叠 亚单位。蛋白质运输需要脂质囊泡形成，该过程依赖于脂质翻转酶活性 建立双层的两个小叶之间的成分不对称性。对此，我们有 确定了所有三类酵母脂质翻转酶的结构。这项续订提案延续了我们的 总体目标是了解蛋白质糖基化和运输。我们建议解决两个具体问题 知识差距：两种蛋白质甘露糖基转移酶的结构和机制，以及最近如何 发现三元蛋白质复合物 Arl1-Gea2-Drs2 将脂质翻转活性与膜曲率耦合起来 形成，从而促进蛋白质和膜运输的下游囊泡出芽过程。 Drs2 是形成 AP-1/网格蛋白包被囊泡所需的磷脂酰丝氨酸翻转酶，该囊泡可移动 在跨高尔基体网络（TGN）和早期内体之间来回移动。小 GTPase Arl1 是 ADP-核糖基化因子 (Arf) 家族的成员，由 Arf 鸟嘌呤核苷酸交换激活 因子 Gea2。 Arl1 专门在 TGN 中运营，是 Arf 家族中最不为人所知的成员。 我们将在体外重建三元复合物并进行全面的结构功能研究。蛋白质 糖基化和运输与肿瘤发生和癌症进展密切相关。我们的机械 研究将填补这些基础知识空白，我们的衍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.