The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Abstract

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the We propose that HOPS localizes via the Vps41p ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.

Multivalent Rab interactions determine tether-mediated membrane fusion

“…Although these studies agree with the bridging of Ypt7-decorated membranes, the direct involvement of the Rab-specific subunits was not addressed. A recent study that was published while this work was under review showed that a HOPS complex lacking Vps39 fails to tether Ypt7-decorated liposomes (Ho and Stroupe, 2016). Although we agree with the general conclusion, our study extends this by identifying both Rab-binding domains in HOPS, their relative affinity for Ypt7, and their role in fusion.…”

“…It indeed binds highly curved vesicles (Cabrera et al. , 2010) and thus promotes tethering (Ho and Stroupe, 2016). With less-curved vesicles, we did not observe ALPS-dependent tethering in the absence of Ypt7, and HOPS lacking the motif promoted fusion like wild-type HOPS (this study).…”

How and why intralumenal membrane fragments form during vacuolar lysosome fusion

“…Concomitantly, active Ypt7 recruits its cognate effector complex HOPS to this site, which could also contribute to stalk formation through two mechanisms: Vps41, a Ypt7 effector protein within the HOPS complex, contains an amphipathic lipid-packing sensor (ALPS) domain that inserts itself into the outer leaflet of the membrane at the vertex ring (Cabrera et al , 2010). In addition to stabilizing HOPS on membranes (Ho and Stroupe, 2016), it could also deform the outer leaflets in a way that promotes stalk formation. Vps33, an SM protein within HOPS, may also contribute to lipid mixing.…”

Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering

“…Although Rab-family small GTPases and specific Rab-interacting effector proteins have generally been recognized as the conserved protein families responsible for membrane tethering [18,19], the mechanisms by which these key components directly and physically act upon the tethering process remain elusive [8,20]. Using a chemically defined reconstitution system with purified protein components and synthetic liposomal membranes, recent biochemical studies have uncovered a handful of protein machineries that directly trigger membrane tethering, including the human Golgi coiled-coil protein GMAP-210 (Golgi microtubuleassociated protein 210) [21], the human endosomal coiled-coil protein EEA1 (early endosome antigen 1) [22], the yeast heterohexameric HOPS (homotypic vacuole fusion and vacuole protein sorting) tethering complex [23][24][25], and Rab-family small GTPases in yeast [26] and humans [8,27,28]. Although the experimental data obtained from the chemically defined systems provide novel mechanistic insights into the molecular functions of these putative membrane tethers [8,[21][22][23][24][25][26][27][28], an important caveat is that the reconstituted tethering reactions observed in vitro can adequately recapitulate membrane tethering events that occur between the membranebounded carriers and subcellular membrane compartments in vivo, particularly in terms of specificity and efficiency [8].…”

“…Using a chemically defined reconstitution system with purified protein components and synthetic liposomal membranes, recent biochemical studies have uncovered a handful of protein machineries that directly trigger membrane tethering, including the human Golgi coiled-coil protein GMAP-210 (Golgi microtubuleassociated protein 210) [21], the human endosomal coiled-coil protein EEA1 (early endosome antigen 1) [22], the yeast heterohexameric HOPS (homotypic vacuole fusion and vacuole protein sorting) tethering complex [23][24][25], and Rab-family small GTPases in yeast [26] and humans [8,27,28]. Although the experimental data obtained from the chemically defined systems provide novel mechanistic insights into the molecular functions of these putative membrane tethers [8,[21][22][23][24][25][26][27][28], an important caveat is that the reconstituted tethering reactions observed in vitro can adequately recapitulate membrane tethering events that occur between the membranebounded carriers and subcellular membrane compartments in vivo, particularly in terms of specificity and efficiency [8]. In this study, to further explore the physiological significance of reconstituted Rab-mediated membrane tethering reactions [8,27,28], we comprehensively and quantitatively evaluated the intrinsic tethering potency of representative human Rab-family small GTPases to directly and physically link the two distinct lipid bilayers together via trans-assembly of membraneanchored Rab proteins in homotypic and heterotypic arrangements.…”