Human Rab small GTPase- and class V myosin-mediated membrane tethering in a chemically defined reconstitution system

Abstract

Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions.

Keywords: Myo5; Rab; liposome; membrane reconstitution; membrane tethering; membrane trafficking; myosin; small GTPase.

Figure 1.

Purification of human Rab GTPases used in this reconstitution study. A, schematic representation of the endosomal Rabs (Rab4a, Rab5a, Rab7a, Rab9a, Rab11a, and Rab14), non-endosomal Rabs (Rab1a and Rab3a), and HRas GTPase in humans showing their amino acid residues, domains (Ras-superfamily GTPase domains and hypervariable regions), and intracellular locations, which include early endosome (EE), recycling endosome (RE), plasma membrane (PM), late endosome (LE), lysosome (Ly), ER, Golgi, and secretory vesicle (SV). B, Coomassie Blue-stained gels of purified recombinant proteins of the C-terminally His₁₂-tagged endosomal Rab, non-endosomal Rab, and HRas GTPases and the untagged form of Rab11a used in this study. C, intrinsic GTP hydrolysis activities of purified Rab proteins. Purified Rab-His₁₂, HRas-His₁₂, and untagged Rab11a proteins (2 μm) were incubated at 30 °C for 1 h in RB150 containing MgCl₂ (6 mm), DTT (1 mm), and GTP (1 mm) or GTPγS (1 mm) for the control followed by an assay of the released free phosphate molecules using a malachite green-based reagent.

Figure 2.

Endosomal Rab GTPases directly initiate membrane tethering by themselves in a chemically defined reconstitution system. A, schematic representation of liposome turbidity assays for testing Rab-mediated liposome tethering (shown in B–G). B–G, endosomal Rab-His₁₂ proteins (each at 0.5–4 μm), Rab4a-His₁₂ (B), Rab5a-His₁₂ (C), Rab7a-His₁₂ (D), Rab9a-His₁₂ (E), Rab11a-His₁₂ (F), and Rab14-His₁₂ (G), were incubated with synthetic liposomes bearing physiological mimic lipid composition (400 nm diameter, 0.5 mm lipids) in RB150 containing MgCl₂ (5 mm) and DTT (1 mm) at room temperature for 300 s. During incubation, turbidity changes in the Rab-liposome–mixed reactions were monitored by measuring the absorbance at 400 nm. The protein-to-lipid molar ratios used for these turbidity reactions were from 1:1000 to 1:125 as indicated.

Figure 3.

Non-endosomal Rab GTPases have the inherent potency to specifically mediate membrane tethering. A, schematic representation of liposome turbidity assays for the non-Rab Ras-superfamily GTPase, HRas, and non-endosomal Rab GTPases, Rab1a in ER-Golgi traffic and Rab3a in exocytosis (shown in B–D). B–D, liposome turbidity assays were employed as described in the legend for Fig. 2, B–G, with HRas-His₁₂ (B), Rab1a-His₁₂ (C), and Rab3a-His₁₂ (D) proteins (each at 0.5–4 μm in final) and physiological mimic synthetic liposomes (0.5 mm total lipids in final). The protein-to-lipid molar ratios used are indicated.

Figure 4.

Membrane association of Rab-His₁₂ proteins onto DOGS-NTA–bearing liposomes. A, schematic representation of liposome co-sedimentation assays for testing membrane attachment of the Rab-His₁₂ proteins used in Figs. 2 and 3. B–K, Rh-labeled liposomes (400 nm diameter, 1 mm total lipids in final) were incubated (30 °C, 30 min) with Rab4a-His₁₂ (B), Rab5a-His₁₂ (C), Rab7a-His₁₂ (D), Rab9a-His₁₂ (E), Rab11a-His₁₂ (F), Rab14-His₁₂ (G), Rab1a-His₁₂ (H), Rab3a-His₁₂ (I), HRas-His₁₂ (J), and BSA for a negative control (K) (2 μm final for each) and ultracentrifuged (50,000 rpm, 30 min, 4 °C). The supernatants (sup) and precipitates (ppt) obtained were analyzed by SDS-PAGE and Coomassie Blue staining.

Figure 5.

Rab-mediated membrane tethering induces the formation of massive liposome clusters. A, schematic representation of fluorescence microscopic observations of Rab-mediated liposome clusters. B–U, fluorescence images (B, D, F, H, J, L, N, P, R, and T) and bright-field images (C, E, G, I, K, M, O, Q, S, and U) of Rab-mediated liposome clusters. Fluorescently labeled liposomes bearing Rh-PE (1000 nm diameter, 0.5 mm lipids in final) were incubated at 30 °C for 1 h in the absence (B and C) and presence of the Rab- and Ras-family GTPases (4 μm each in final), including Rab4a-His₁₂ (D and E), Rab5a-His₁₂ (F and G), Rab7a-His₁₂ (H and I), Rab9a-His₁₂ (J and K), Rab11a-His₁₂ (L and M), Rab14-His₁₂ (N and O), Rab1a-His₁₂ (P and Q), Rab3a-His₁₂ (R and S), and HRas-His₁₂ (T and U), and subjected to fluorescence microscopy. Scale bars: 20 μm.

Figure 6.

Particle size distributions of liposome clusters induced by Rab-mediated membrane tethering. A, particle sizes of the Rab-mediated liposome clusters observed in the fluorescence images shown in Fig. 5. B, particle numbers and average sizes (μm²) of the Rab-mediated liposome clusters observed in the fluorescence images shown in Fig. 5.

Figure 7.

Class V myosin globular tail domains, Myo5A-GTD and Myo5B-GTD, strongly stimulate Rab11a-dependent membrane tethering. A, schematic representation of class V myosins in humans, Myo5A and Myo5B, showing their amino acid residues and domains including myosin motor domains, IQ motifs, coiled-coil regions, and GTDs. Representative Myo5-interacting Rab GTPases and the Rab-binding regions in Myo5A and Myo5B are indicated. B, the Coomassie Blue-stained gel of purified Myo5A-GTD and Myo5B-GTD proteins, which are comprised of amino acid residues 1534–1855 and 1526–1848, respectively. C, schematic representation of liposome turbidity assays for testing Rab11a- and Myo5-GTD–dependent liposome tethering (shown in D and E). D and E, liposome turbidity assays were employed with Rab11a-His₁₂ (1 μm final) as described in the legend for Fig. 2F but in the presence of Myo5A-GTD (D) and Myo5B-GTD (E) (0.5–2 μm final). F–K, fluorescence images of Rab11a-mediated liposome clusters in the presence of Myo5-GTDs. Rab11a-His₁₂ (3 μm final) and Myo5A-GTD or Myo5B-GTD (3 μm final) were preincubated at 30 °C for 30 min, mixed with Rh-labeled liposomes (1000 nm diameter, 0.8 mm lipids in final), incubated further (30 °C, 30 min), and subjected to fluorescence microscopy (J and K). For a control, Rab11a-His₁₂, Myo5-GTD, or both were omitted from the reactions where indicated (F–I). Scale bars: 20 μm.

Figure 8.

Guanine nucleotide dependence of Rab11a-mediated membrane tethering in the presence of Myo5A-GTD and Myo5B-GTD. A, schematic representation of liposome turbidity assays for testing Rab11a- and Myo5-GTD–dependent liposome tethering in the presence of GTP (shown in B–G). B and C, Rab11a/Myo5-dependent membrane tethering is strongly and specifically promoted by the addition of GTP. Liposome turbidity assays with Rab11a-His₁₂ (1 μm) and Myo5A-GTD (1 μm) (B) or Myo5B-GTD (2 μm) (C) were performed as described in the legend for Fig. 7, D and E, but in the presence of GTP (1 mm). D and E, liposome turbidity assays were employed with Rab11a-His₁₂ and Myo5A-GTD (D) or Myo5B-GTD (E), as described in B and C, in the presence of various concentrations of GTP (1 μm–1 mm). F and G, liposome turbidity assays were employed with Rab11a-His₁₂ and Myo5A-GTD (F) or Myo5B-GTD (G), as described in B and C, in the presence of GTP, GTPγS, and GDP (each at 1 mm).

Figure 9.

Membrane association of Myo5-GTD proteins with Rab11a-anchored liposomes. A, schematic representation of liposome co-sedimentation assays for testing membrane binding of Myo5A-GTD and Myo5B-GTD to Rab11a-bound liposomes. B–G, liposome co-sedimentation assays were employed as described in the legend for Fig. 4, with Rh-labeled liposomes (400 nm diameter, 1 mm lipids) and Rab11a-His₁₂ (2 μm) (B and E) but in the presence of Myo5A-GTD (2 μm) (B–D), Myo5B-GTD (2 μm) (E–G), and GTP (1 mm). For a control, the reactions without Rab11a-His₁₂ (C and F) or with the untagged form of Rab11a lacking a His₁₂ tag (untagged Rab11a) (D and G) were also tested. The supernatants (sup) and precipitates (ppt) obtained were analyzed by SDS-PAGE and Coomassie Blue staining.

Figure 10.

Myo5A-GTD and Myo5B-GTD selectively activate Rab11a-dependent membrane tethering. A, schematic representation of liposome turbidity assays (shown in B and C) for the various Rab GTPases (Rab11a, Rab1a, Rab4a, Rab9a, and Rab14) in the presence of Myo5-GTDs and GTP. B and C, Myo5-GTDs specifically promote efficient membrane tethering mediated by the cognate Rab GTPase, Rab11a. Liposome turbidity assays were employed using Myo5A-GTD (B) or Myo5B-GTD (C) and GTP, as described in the legend for Fig. 8, B and C, for Rab11a, Rab1a, Rab4a, Rab9a, and Rab14 GTPases.

Figure 11.

Rab11a- and Myo5-GTD–dependent membrane tethering requires the membrane attachment of Rab11a on both of two opposing membranes destined to tether. A, schematic representation of the streptavidin bead–based liposome-tethering assay described in B and C using two types of liposomes bearing either biotin-PE/DOGS-NTA/Rh-PE or FL-PE/DOGS-NTA and purified Rab11a-His₁₂ and Myo5-GTD proteins. B, streptavidin bead–based liposome-tethering assays with Rab11a-His₁₂ and Myo5-GTDs. The biotin-labeled and FL-labeled liposomes were incubated with streptavidin-coated magnetic beads (30 °C, 2 h), mixed with Rab11a-His₁₂ and Myo5A-GTD or Myo5B-GTD, and incubated further (30 °C, 10 min). The FL-labeled liposomes tethered to the biotin-labeled liposomes on streptavidin beads were quantified by measuring the FL fluorescence. For a control, Rab11a, Myo5A-GTD, and Myo5B-GTD were omitted from the reactions where indicated (lanes 1–4). AU, arbitrary units. C, streptavidin bead–based liposome-tethering assays were employed with Rab11a-His₁₂ and Myo5-GTDs as described in B, but FL-labeled liposomes lacking DOGS-NTA were used instead where indicated (lanes 2 and 4). D–G, fluorescence microscopy was performed as described in Fig. 7, F–K, with Rh-labeled liposomes (0.4 mm lipids), FL-labeled liposomes (0.4 mm lipids), Rab11a-His₁₂ (3 μm), and Myo5A-GTD (D and E) or Myo5B-GTD (F and G) (each at 3 μm). Rh-labeled liposomes lacking DOGS-NTA were used instead where indicated (E and G). Scale bars: 10 μm.