Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions

Membrane tethering potency of Rab-family small GTPases is defined by the C-terminal hypervariable regions

10.1101/2020.06.28.176792 ◽

2020 ◽

Author(s):

Sanae Ueda ◽

Naoki Tamura ◽

Joji Mima

Keyword(s):

Lipid Bilayers ◽

Membrane Lipids ◽

Hypervariable Region ◽

Coiled Coil ◽

Small Gtpases ◽

Small Gtpase ◽

Full Length ◽

Rab Gtpases ◽

Rab Family ◽

Membrane Tethering

AbstractMembrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5-to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

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

10.1101/129072 ◽

2017 ◽

Author(s):

Motoki Inoshita ◽

Joji Mima

Keyword(s):

Lipid Bilayers ◽

Membrane Trafficking ◽

Small Gtpases ◽

Small Gtpase ◽

Effector Proteins ◽

Rab Proteins ◽

Dependent Manner ◽

Class V ◽

Rab Family ◽

Membrane Tethering

AbstractMembrane tethering is a fundamental process essential for 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, have been reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes still 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 apposing 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.

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

10.1101/544379 ◽

2019 ◽

Author(s):

Kazuya Segawa ◽

Naoki Tamura ◽

Joji Mima

Keyword(s):

Membrane Trafficking ◽

Coiled Coil ◽

Small Gtpases ◽

Physical Contact ◽

Molar Ratios ◽

Membrane Surfaces ◽

Subcellular Membrane ◽

Rab Family ◽

Membrane Tethering ◽

Protein Components

AbstractMembrane tethering is a highly regulated event that occurs 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, 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 in a chemically defined reconstitution system. 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 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab-to-lipid ratio of 1:3,000. Comprehensive reconstitution studies 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. Thus, our findings establish that, in the physiological context, homotypic and heterotypic trans-assembly of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.

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Reconstitution of membrane tethering mediated by Rab-family small GTPases

Biophysical Reviews ◽

10.1007/s12551-017-0358-3 ◽

2017 ◽

Vol 10 (2) ◽

pp. 543-549 ◽

Cited By ~ 10

Author(s):

Joji Mima

Keyword(s):

Small Gtpases ◽

Rab Family ◽

Membrane Tethering

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.007947 ◽

2019 ◽

Vol 294 (19) ◽

pp. 7722-7739 ◽

Cited By ~ 7

Author(s):

Kazuya Segawa ◽

Naoki Tamura ◽

Joji Mima

Keyword(s):

Small Gtpases ◽

Rab Family ◽

Membrane Tethering

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Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment

The Journal of Cell Biology ◽

10.1083/jcb.137.3.563 ◽

1997 ◽

Vol 137 (3) ◽

pp. 563-580 ◽

Cited By ~ 151

Author(s):

Gregory Jedd ◽

Jon Mulholland ◽

Nava Segev

Keyword(s):

Protein Transport ◽

Small Gtpases ◽

Yeast Cells ◽

Secretory Vesicles ◽

Golgi Compartment ◽

Rab Family ◽

Vacuolar Protein ◽

Exocytic Pathway ◽

Mutant Cells

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.

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Rab GTPases in Osteoclastic Endomembrane Systems

BioMed Research International ◽

10.1155/2018/4541538 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 3

Author(s):

Michèle Roy ◽

Sophie Roux

Keyword(s):

Plasma Membrane ◽

Small Gtpases ◽

Vesicular Trafficking ◽

Degradation Pathways ◽

Rab Gtpases ◽

Bone Homeostasis ◽

Rab Family ◽

Autophagic Degradation

Osteoclasts (OCs) are bone-resorbing cells that maintain bone homeostasis. OC differentiation, survival, and activity are regulated by numerous small GTPases, including those of the Rab family, which are involved in plasma membrane delivery and lysosomal and autophagic degradation pathways. In resorbing OCs, polarized vesicular trafficking pathways also result in formation of the ruffled membrane, the resorbing organelle, and in transcytosis.

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