Abstract
Membrane tethering is a highly regulated event 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/lipid molar ratios of 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab/lipid ratio of 1:3,000. 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.
Highlights
Membrane tethering is a highly regulated event 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
A large body of earlier genetic and biochemical studies have reported that Rab (Ras-related in brain)-family small GTPases and specific sets of Rab-interacting proteins (i.e. Rab effectors), such as the coiled-coil tethering proteins and multisubunit tethering complexes, participate in the membrane tethering process [2,3,4,5,6,7,8,9,10,11] that occurs prior to the membrane docking and fusion events mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)2-family proteins and SNARE-interacting chaperones [12, 13], which are additional critical steps to confer fidelity to the membrane-trafficking process [12,13,14,15,16,17]
They all share a typical structural feature of the Rab small GTPase family and are known to be small monomeric proteins of around 25 kDa that consist of an N-terminal nonconserved flexible segment, a conserved globular Ras-superfamily GTPase domain (G-domain) in the middle, a C-terminal flexible hypervariable region (HVR), and one or two geranylgeranyl lipid anchors, which are post-translationally conjugated to the C terminus of the HVR (Fig. 1A and Fig. S1) [8, 31, 32]
Summary
From over 60 protein isoforms of Rab-family small GTPases in human cells, which constitute the largest branch of the Ras superfamily [29], we selected 10 representative human Rabfamily proteins (Rab1a, Rab3a, Rab4a, Rab5a, Rab6a, Rab7a, Rab9a, Rab11a, Rab27a, and Rab33b; Fig. 1 and Fig. S1) to assess intrinsic membrane tethering capacities in this reconstitution study. When comparing the two early endosomal Rab isoforms, Rab4a and Rab5a, there were over 20and 300-fold differences in the average particle size and total area of particles, respectively (Table 1) These quantitative imaging data further establish that there is a wide diversity in the intrinsic tethering potency among the human Rab-family proteins (Fig. 4 and Table 1), even though all of the Rab isoforms share the conserved Ras-superfamily GTPase domain as a major portion in their amino acid sequences (Fig. 1A and Fig. S1) and exhibit comparable GTP hydrolysis activities (Fig. 2A). Particle sizes of the Rab-induced liposome clusters observed in fluorescence microscopic images were quantitatively measured using ImageJ2 software
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