Summary
Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Paradoxically, genetic studies placed Uso1 acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S substitutions in the GHD permitting growth of otherwise inviable rab1-deficient Aspergillus nidulans. Remarkably, the double mutant suppresses the complete absence of RAB1. Full-length Uso1 and CTRΔ proteins are dimeric and the GHD lacking the CC/CTR is monomeric irrespective of whether they carry or not E6K/G540S. Microscopy showed recurrence of Uso1 on puncta (60 sec half-life) colocalizing with RAB1 and less so with early Golgi markers Sed5 and GeaA/Gea1/Gea2. Localization of Uso1 but not of Uso1E6K/G540S to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. By S-tag-coprecipitation we demonstrate that Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Bos1 and Bet1 bind the Uso1 GHD directly, but Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. AlphaFold2 predicts that G540S actually increases binding of GHD to the Bos1 Habc domain. In contrast, E6K seemingly increases membrane targeting of an N-terminal amphipathic α-helix, explaining phenotypic additivity. Overexpression of E6K/G540S and wild-type GHD complemented uso1Δ. Thus, a GHD monomer provides the essential Uso1 functions, demonstrating that long-range tethering activity is dispensable. Therefore, when enhanced by E6K/G540S, Uso1 binding to Bos1/Bet1 required to regulate SNAREs bypasses both the contribution of RAB1 to Uso1 recruitment and the reported role of RAB1 in SNARE complex formation (Lupashin and Waters, 1997), suggesting that the latter is consequence of the former.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
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