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bioRxiv · 10.1101/2022.05.24.493304

STX5's flexibility in SNARE paring supports Golgi functions

D'Souza, Z.·Pokrovskaya, I. D.·Lupashin, V.
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Abstract

The intracellular transport system is an evolutionally conserved, essential, and highly regulated network of organelles and transport vesicles that traffic protein and lipid cargoes within the cell. The events of vesicle formation, budding and fusion are orchestrated by the trafficking machinery - an elaborate set of proteins including small GTPases, vesicular coats, tethers, and SNAREs. The Golgi - the central organelle in this transport network, receives, modifies and sorts secretory and endocytic cargo. Glycosylation is one of the major modifications that occur within the Golgi, which houses enzymes and other components of glycosylation machinery. According to the current Golgi maturation model, Golgi resident proteins are constantly recycled from the late (trans) Golgi compartments to the early compartment (cis) by the evolutionary conserved vesicular trafficking machinery. The key modulator of vesicular trafficking and glycosylation at the Golgi is the Conserved Oligomeric Golgi (COG) complex - its interaction vesicular trafficking machinery particularly Golgi SNAREs (STX5, GS28 (GOSR1), GS15 (BET1L) and YKT6) that drive fusion of incoming vesicles. Since the COG complex functions upstream of SNARE-mediated vesicle fusion, we hypothesize that depletion of Golgi v-SNAREs would mirror defects observed in COG deficient cells. To test this, we created single and double knockouts (KO) of GS28 and GS15 in HEK293T cells and analyzed 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. Quantitative MS analysis of STX5-interacting SNAREs revealed unexpected flexibility of Golgi SNARE pairing in mammalian cells. We uncovered two novel non-canonical Golgi SNARE complexes - STX5/VTI1B/GS15/YKT6 and STX5/SNAP29/VAMP7 which were upregulated in GS28 KO cells. Analysis of cells co-depleted for GS28/SNAP29 or GS28/VTI1B SNAREs revealed escalated defects in Golgi glycosylation, indicating that upregulation of these complexes functionally substitutes deleted GS28. Our data points to the remarkable plasticity in the intra-Golgi membrane fusion machinery which is controlled by the COG complex.

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D'Souza, Z., Pokrovskaya, I. D., Lupashin, V.. 2022-05-25. STX5's flexibility in SNARE paring supports Golgi functions. https://doi.org/10.1101/2022.05.24.493304

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