Elucidating the Role of GRASPs In and Out of the Golgi Apparatus
Aihaiti, Yierpanijiang
2021
Abstract
The stacking of flattened cisternae is the basic structure of the Golgi apparatus. In mammalian cells, these stacks are linked into a ribbon-like structure located at the perinuclear region of the cell. It has been previously demonstrated that the two Golgi peripheral membrane proteins, GRASP55 and GRASP65, form trans-oligomers to link flattened cisternae into stacks. Depletion of either one of the two GRASP proteins decreases the number and length of the cisternae in each stack, whereas depletion of both GRASPs causes complete unstacking and ribbon unlinking of the Golgi. Functional studies revealed that Golgi destruction by GRASP-depletion accelerates protein trafficking in the Golgi but results in N-glycosylation defects of cell surface proteins and mis-sorting lysosome enzymes. However, whether these defects affect cellular functions such as cell attachment, migration and growth was unclear. In the first part of my thesis (Chapter 2), we disrupted the Golgi stacks by GRASP55/65 knockdown/knockout and determined the subsequent impact on multiple cellular activities. We found that this decreases cell attachment, migration, and invasion mainly due to the reduction of α5β1 integrin levels. Our results showed that GRASP depletion decreases α5β1integrin via reducing its synthesis but not due to accelerated degradation. We also found that GRASP depletion accelerates cell growth, which may be attributed to the increased overall protein synthesis and accelerated protein trafficking. The Golgi is well-known for its unique stacked structure, but why stack formation is important for Golgi function is a long-standing question in cell biology. To study the significance of Golgi stack formation in glycosaminoglycan (GAGs) synthesis, in Chapter 3 of my thesis, we determined the effect of Golgi unstacking on the synthesis, sulfation, and secretion of heparan sulfate (HS) and chondroitin sulfate (CS), two major types of GAGs synthesized in the Golgi. We found that GRASP depletion increases HS synthesis while decreasing CS synthesis in cells, alters HS and CS sulfation, and reduces both HS and CS secretion. We identified EXTL3, GalNacT1, and PAPSS2, key enzymes in HS and CS synthesis and sulfation pathways, whose level changes cause the alterations in GAG synthesis and sulfation. In addition to its roles in Golgi structure formation, GRASP55 has recently been shown to regulate autophagy and unconventional secretion via translocation to other membrane structures upon stress conditions. Although the function of GRASP55 in autophagy and unconventional secretion seems to be interconnected, the mechanism of GRASP55-dependent unconventional secretion is poorly understood. In the third part of my thesis (Chapter 4), we determined the role of GRASP55 in unconventional secretion of cytosolic neurotoxic proteins using polyQ-Htt as a marker. We discovered that polyQ-Htt is unconventionally secreted via a GRASP55-dependent pathway, which regulates its aggregation and toxicity. GRASP55 regulates the secretion of Htt via two mechanisms: 1) facilitation of autophagosome-lysosome fusion by functioning as a tether between LC3 on autophagosomes and LAMP2 on lysosomes; 2) interacting with and stabilization of p23/TMED10, an ER-Golgi intermediate compartment (ERGIC) protein that functions as a channel for cytoplasmic cargoes translocation into ERGIC lumen. To further elucidate the role of GRASP55 in unconventional secretion, we performed secretome analysis to systematically analyze GRASP55-dependent conventional and unconventional cargoes and identified new bona fide unconventional pathway cargoes such as TAGLN1, PAICS, and PRDX1. Our research sheds light on the understanding of the unconventional protein secretory pathway and progression of Huntington’s disease.Deep Blue DOI
Subjects
Golgi unconventional secretion autophagy Huntington's Disease (HD) and huntingtin protein GRASP55 integrin
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