PT - JOURNAL ARTICLE AU - Kazuya Segawa AU - Naoki Tamura AU - Joji Mima TI - Homotypic and heterotypic <em>trans-assembly</em> of human Rab-family small GTPases in reconstituted membrane tethering AID - 10.1101/544379 DP - 2019 Jan 01 TA - bioRxiv PG - 544379 4099 - http://biorxiv.org/content/early/2019/03/05/544379.short 4100 - http://biorxiv.org/content/early/2019/03/05/544379.full AB - Membrane tethering is a highly regulated event that occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g., Rab-to-lipid molar ratios of 1:100-1:3000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab-to-lipid ratio of 1:3000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.SNAREsoluble N-ethylmaleimide-sensitive factor attachment protein receptorHVRhypervariable regionPCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineRhrhodamineHRVhuman rhinovirusFLfluoresceinPO1-palmitoyl-2-oleoyl