Dataset Information

Syntaxin5’s flexibility in SNARE pairing supports Golgi functions

ABSTRACT: The intracellular transport system is an evolutionally conserved, essential, and highly regulated network of organelles and small transport carriers that traffic protein and lipid cargoes within the cell. The Conserved Oligomeric Golgi (COG) complex is the major Golgi Multisubunit Tethering Complex. Its key function is orchestration of SNARE mediated fusion of cargo carriers at the Golgi. We hypothesized the depletion of the major Golgi SNAREs GS28 (GOSR1) and GS15 (BET1L) due to COG malfunction is the major contributor to COG-related glycosylation defects. To test this, we created single, double and triple knockouts (KO) of Golgi SNAREs in HEK293T cells and analyzed the resulting mutants using a comprehensive set of biochemical, mass-spectrometry (MS) and microscopy approaches. Deletion of GS28 significantly affected GS15, but not the other two partners, STX5 and YKT6. Surprisingly, our analysis revealed that COG dysfunction is more deleterious for Golgi function than disrupting the canonical Golgi SNARE complex indicating the existence of an adaption mechanism. Indeed, quantitative mass-spectrometry analysis of STX5-interacting proteins revealed unexpected flexibility in Golgi SNARE pairing in mammalian cells. We uncovered two novel non-canonical Golgi SNARE complexes – STX5/SNAP29/VAMP7 and STX5/VTI1B/GS15/YKT6 which were increased in GS28 KO cells. Upon GS28 deletion, SNAP29 remarkably localized to the Golgi. Merely mislocalizing STX5 from Golgi abolished the SNARE substitution causing dramatic glycosylation defects. Our data points to the remarkable plasticity in the intra-Golgi membrane fusion machinery and places STX5 as the only essential Golgi Qa-SNARE.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

SUBMITTER: Zinia DSouza

LAB HEAD: Vladimir V Lupashin

PROVIDER: PXD036741 | Pride | 2023-06-26

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	Lupashin_102620_Sample_01.dia.elib	Other
	Lupashin_102620_Sample_01.raw	Raw
	Lupashin_102620_Sample_02.dia.elib	Other
	Lupashin_102620_Sample_02.raw	Raw
	Lupashin_102620_Sample_03.dia.elib	Other

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Syntaxin-5's flexibility in SNARE pairing supports Golgi functions.

D'Souza Zinia Z Pokrovskaya Irina I Lupashin Vladimir V VV

Traffic (Copenhagen, Denmark) 20230621 8

Deficiency in the conserved oligomeric Golgi (COG) complex that orchestrates SNARE-mediated tethering/fusion of vesicles that recycle the Golgi's glycosylation machinery results in severe glycosylation defects. Although two major Golgi v-SNAREs, GS28/GOSR1, and GS15/BET1L, are depleted in COG-deficient cells, the complete knockout of GS28 and GS15 only modestly affects Golgi glycosylation, indicating the existence of an adaptation mechanism in Golgi SNARE. Indeed, quantitative mass-spectrometry ...[more]

PMID: 37340984

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Project description:Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.