Gatekeeper helix activates Golgi SM protein Sly1 and directly mediates close-range vesicle tethering

Abstract

c. 160 words)The essential Golgi protein Sly1 is a member of the SM (Sec1/mammalian Unc-18) family of SNARE chaperones. Sly1 was originally identified through gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 5 family members is not only autoinhibitory, but also acts as a positive effector. An amphipathic helix within the loop directly binds high-curvature membranes; membrane binding is required for relief of Sly1 autoinhibition and allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 allele bypasses requirements for diverse tethering factors but loses this functionality if Sly1 membrane binding is impaired. We propose that 10 long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to activate SNARE assembly and fusion in the early secretory pathway. 25 2010; Rizo and Sudhof, 2012; Sudhof and Rothman, 2009) but biochemical work, structural studies, and single-molecule force spectroscopy suggest that SM proteins are assembly chaperones for trans-SNARE complex formation, and that SMs act, at least in part, by templating the initial SNARE zippering reaction (Baker et al., 2015;Jiao et al., 2018) and by protecting appropriately formed prefusion complexes from kinetic proofreading by the SNARE 30

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

“…, 2019 ; Duan et al. , 2020a , 2020b ). Exogenous overexpression of an SCFD1-HaloTag fusion colocalized with endogenous ACBD3 at the cis -Golgi apparatus ( Figure 3A ).…”

“…It has been proposed that SCFD1 interacts with the cis -Golgi SNARE STX5 which promotes the fusion of the incoming COPII vesicles by forming a SNARE complex with the v-SNARE SEC22B ( Duan et al. , 2020a , 2020b ). Interestingly, we also identified SEC22B as a binding partner of ACBD3 in our mass spectrometry experiment ( Figure 2A ).…”

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

“…SCFD1 (aka SLY1) is a soluble cytosolic Sec1/Munc18-like protein recruited to the cis-Golgi apparatus to participate in catalysing the formation of a SNARE complex [45][46][47] . Exogenous overexpression of an SCFD1-HaloTag fusion colocalised with endogenous ACBD3 at the cis-Golgi apparatus (Figure 3A).…”

“…Thus, SCFD1 is upstream of ACBD3, which is upstream of PI4KIIIβ. It has been proposed that SCFD1 interacts with the cis-Golgi SNARE STX5 allowing it to switch to an open conformation by exposing its SNARE domain 46,47 . Then, SCFD1 bridges STX5 to the incoming COPII vesicles and promotes the formation of the SNARE complex between STX5 and the v-SNARE SEC22B, ultimately leading to membrane fusion.…”