Analysis of the Molecular Mechanism and Physiological Roles of the GRASP Proteins in the Golgi Apparatus and Autophagy

Golgi Apparatus;GRASP55/65;Autophagy;CRISPR;PI3K complex;Trafficking;Molecular;Cellular and Developmental Biology;Science;Molecular, Cellular, and Developmental Biology

Wang, LeibinXu, Haoxing ;

University of Michigan

关键词: Golgi Apparatus;    GRASP55/65;    Autophagy;    CRISPR;    PI3K complex;    Trafficking;    Molecular;    Cellular and Developmental Biology;    Science;    Molecular, Cellular, and Developmental Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/147487/leibinw_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The Golgi apparatus resides at the center of the secretory pathway and receives almost the entire output of the endoplasmic reticulum (ER). Within the Golgi, proteins and lipids undergo multiple modifications, including N- or O-linked glycosylation, phosphorylation and proteolytic cleavage. After defined modifications, mature cargo proteins and lipids are sorted into secretory vesicles for transport to the plasma membrane, endosomes and lysosomes, or outside of the cell. In most eukaryotic cells, the Golgi apparatus is composed of stacks of tightly aligned flattened cisternae, which are laterally linked into a ribbon like structure located in the perinuclear region. The Golgi reassembly stacking proteins of 65 kDaand 55 kDa (GRASP65 and GRASP55) were two proteins originally identified as Golgi stacking factors. GRASP65 and GRASP55 localize to the cis- and medial-trans-cisternae, respectively, where they form homo-dimers and trans-oligomers to hold adjacent cisternae into a stack. Moreover, GRASP55 is also involved in unconventional secretion and autophagy. To further understand GRASPs’ functions, my research focused on the following two questions: 1) How do GRASP proteins play a role in Golgi structure and function? 2) How does GRASP55 regulate autophagy? To answer the first question, in collaboration with others in the lab, I applied the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to knock out GRASP55 and GRASP65, individually or in combination, in HeLa and HEK293 cells. In this study, we showed that double knockout of GRASP proteins disperses the Golgi stacks into single cisternae and tubulovesicular structures, accelerates protein trafficking, and impairs accurate glycosylation of proteins and lipids. These results demonstrate a critical role for GRASPs in maintaining the stacked structure of the Golgi, and confirmed the hypothesis that a well stacked Golgi is required for accurate post-translational modifications. Additionally, the GRASP knockout cell lines developed in this study will be useful tools for studying the role of GRASP proteins in other important cellular processes.To answer the second question, I firstly induced autophagy by nutrient deprivation and determined the effects on the Golgi. I found that upon amino acid starvation, trans-Golgi derived membrane fragments colocalize with autophagosomes. Depletion of GRASP55, but not GRASP65, increases both LC3-II and p62 levels. Further studies demonstrated that upon amino acid starvation, GRASP55 facilitates autophagosome-lysosome fusion through two mechanisms, one is by physically tethering autophagosomes and lysosomes through the interactions with LC3 on autophagosomes and LAMP2 on late endosomes/lysosomes, and the other is by interacting with Beclin 1, UVRAG, Vps34 and Bif-1 to facilitate the assembly and membrane association of this phosphoinositide 3-kinase (PI3K) complex. These findings indicate that GRASP55 plays an important role in autophagosome maturation during amino acid starvation.In conclusion, during my PhD research, I generated GRASP55 and GRASP65 knockout cells and confirmed their roles in Golgi structure and function; I also discovered the molecular mechanism of GRASP55’s role in autophagy: as a tether between autophagosome and lysosome, and as a regulator of the PI3K-UVRAG complex. My thesis work proposed a novel target for treatment of Golgi and autophagy related diseases.