The G-Protein Rab5A Activates VPS34 Complex II, a Class III PI3K, by a Dual Regulatory Mechanism



Thomas C. Buckles1, Yohei Ohashi2, Shirley Tremel2, Stephen H. McLaughlin2, Els Pardon3,4, Jan Steyaert3,4, Moshe T.Gordon1, Roger L.Williams2, Joseph J.Falke1


  1. Molecular Biophysics Program, Department of Biochemistry, University of Colorado, Boulder, Colorado
  2. Medical Research Council, Laboratory of Molecular Biology Cambridge University, Cambridge, United Kingdom
  3. VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium
  4. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium


VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.

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VPS34CII and Rab5A are both linked to multiple human diseases. It is also found that a net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily, during endocytosis and early endosome development. To understand the molecular level mechanism by which Rab5A activates lipid kinase activity, two general mechanistic models are proposed in this research using an in vitro single-molecule approach. In these models, it’s known that 13kDa nanobodies CA12588 and CA12588-Cys bind to VPS34CI and VPS34CII. To discover the complex specific nanobodies, a CovalX K200 stabilization kit was first used as a cross-linked agent with purified human VPS34 complexes I and II, and injected into two llamas. At the end stage, enzyme-linked immunosorbent assay was used to discover 21 families and 19 families for complex I and II respectively. HDX-MS experiments were also used to express the CA12588 nanobody.


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