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

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 coiledcoil 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.

Curvature‐sensitive trans‐assembly of human Atg8‐family proteins in autophagy‐related membrane tethering

“…LC3B‐His12 and GATE‐16‐His12 proteins triggered efficient tethering of synthetic, but physiologically mimicking, DOGS‐NTA‐bearing liposomes (100 nm in diameter; Figure a) even at the protein‐to‐lipid molar ratios of 1:1,000 or below (black and blue circles, respectively, in Figure b). However, Rab5a‐His12, which has been so far found to be the most active membrane tether among human Rab‐family small GTPase isoforms, had the substantially less tethering capacity under the same conditions (green circles, Figure b) and, H‐Ras‐His12, used as the negative control of a C‐terminally His12‐tagged protein, was completely unable to initiate liposome tethering in the turbidity assays (cyan circles, Figure b). Moreover, the kinetic liposome turbidity assays indicated that the initial velocities of human LC3B‐ and GATE‐16‐mediated membrane tethering reactions were much higher than that of the Rab5a‐mediated reaction, when tested at the protein‐to‐lipid ratios of 1:500 (Figure c).…”

“…To comprehensively and quantitatively evaluate the intrinsic membrane tethering capacities of human LC3B and GATE‐16 proteins in a chemically defined proteoliposomal system, we employed three types of reconstituted liposome tethering assays, which had been previously developed to test membrane tethering driven by human Rab‐family small GTPases in our recent studies, including a high‐throughput 384‐well microplate‐based endpoint assay to measure the turbidity of liposomes (Figure b), a kinetic assay to monitor liposome turbidity changes (Figure c), and a fluorescence cell counter‐based imaging assay giving an unbiased quantitative data of clusters of tethered liposomes (Figure d,e). Strikingly, both of the human Atg8 proteins exhibited the abilities to drive highly efficient and rapid membrane tethering by themselves, in the absence of any other additional protein components (Figure b–e).…”

“…In the endpoint turbidity assays, we tested the intrinsic membrane tethering potency of LC3B and GATE‐16 proteins comprehensively at the protein‐to‐lipid molar ratios ranging from 1:10,000 to 1:100 (mol/mol), demonstrating that both of the human Atg8‐family proteins can trigger membrane tethering at the protein‐to‐lipid ratio of 1:5,000 and they drive membrane tethering more efficiently at the higher ratios up to 1:100 (black and blue circles, Figure b). Since it has been described that protein densities of fusogens or tethers on membrane surfaces are critical to establishing the physiologically relevant reconstitution systems for protein‐driven membrane tethering and fusion mediated by SNARE‐family proteins and Rab‐family small GTPases, we, therefore, assessed whether the Atg8 protein densities tested in the current tethering assays (Figure b) are relevant to the physiological environments of native Atg8‐family proteins associated with autophagic membrane compartments. Previous works by Klionsky and colleagues quantitatively estimated the protein stoichiometry of several representative Atg proteins at the phagophore assembly site (PAS) by a fluorescence microscopic analysis and also the size of the fully expanded phagophore by an electron microscopic analysis of autophagic bodies in the Pep4p‐deficient yeast vacuoles .…”

“…Four types of DOGS‐NTA‐bearing liposomes were prepared by extrusion through 50‐, 100‐, 200‐, and 400‐nm pore sizes (Figure a) and evaluated for the size distributions using dynamic light scattering, establishing that the liposome preparations had the mean diameters roughly similar to the theoretical pore sizes used and that their size distributions were significantly different from each other (Figure b). Using these 50‐, 100‐, 200‐, and 400‐nm‐diameter liposome preparations (Figure a,b), we employed the kinetic turbidity assays for LC3B, GATE‐16, and Rab5a that is a putative membrane tether functioning in non‐autophagic, endocytic trafficking pathways, followed by determining their maximum tethering capacities and initial tethering velocities from curve fitting of the kinetic data obtained (Figure c‐f).…”

“…After centrifugation of the bead suspensions (15,300 g , 10 min, 4°C), protein concentrations of purified LC3B‐His12 and GATE‐16‐His12 proteins in the supernatants obtained (around 4 ml each) were determined using Protein Assay CBB Solution (Nacalai Tesque) and bovine serum albumin (BSA) for a standard protein. Recombinant proteins of human Rab5a‐His12 and H‐Ras‐His12, which were used as a control protein in the reconstituted liposome tethering assays, were expressed in E. coli cells and purified as described previously …”