The Sec1/Munc18 protein VPS33B forms a uniquely bidirectional complex with VPS16B.

Loss of function variants of VPS33B and VIPAS39 (encoding VPS16B) are causative for arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, where early lethality of patients indicates that VPS33B and VPS16B play essential cellular roles. VPS33B is a member of the Sec1/Munc18 (SM) protein family, and thus thought to facilitate vesicular fusion via interaction with SNARE complexes, as does its paralog VPS33A in the homotypic fusion and vacuole sorting (HOPS) complex. VPS33B and VPS16B have been shown to associate, but little is known about the composition, structure or function of the VPS33B/VPS16B complex. We show here that human VPS33B/VPS16B is a high molecular weight complex, which we expressed in yeast to obtain material for structural, composition and stability analysis. Circular dichroism data indicate VPS33B/VPS16B has a well-folded α-helical secondary structure, for which size exclusion chromatography-multi angle light scattering revealed a MW of ∼315 kDa. Quantitative immunoblotting indicated the complex has a VPS33B:VPS16B ratio of 2:3. Expression of ARC syndrome-causing VPS33B missense variants showed that L30P disrupts complex formation, but not S243F or H344D. Truncated VPS16B containing amino acids 143-316 was sufficient to form a complex with VPS33B. Small angle X-ray scattering and negative staining electron microscopy revealed a two-lobed shape for VPS33B/VPS16B. Avidin tagging indicated that each lobe contains a VPS33B molecule, and they are oriented in opposite directions. From this we propose a structure for VPS33B/VPS16B that allows the copies of VPS33B at each end to interact with separate SNARE bundles and/or SNAREpins, plus their associated membrane components. Thus our observations reveal the only known potentially bidirectional SM protein complex.