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

J Biol Chem. 2017 Nov 10;292(45):18500-18517. doi: 10.1074/jbc.M117.811356. Epub 2017 Sep 22.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Endosomes / enzymology
  • Endosomes / metabolism*
  • Guanosine Triphosphate / metabolism*
  • Histidine / chemistry
  • Histidine / genetics
  • Histidine / metabolism
  • Humans
  • Intracellular Membranes / chemistry
  • Intracellular Membranes / enzymology
  • Intracellular Membranes / metabolism*
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism
  • Liposomes
  • Lysine / analogs & derivatives
  • Lysine / chemistry
  • Lysine / metabolism
  • Myosin Heavy Chains / chemistry
  • Myosin Heavy Chains / genetics
  • Myosin Heavy Chains / metabolism*
  • Myosin Type V / chemistry
  • Myosin Type V / genetics
  • Myosin Type V / metabolism*
  • Oleic Acids / chemistry
  • Oleic Acids / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Prenylation
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Succinates / chemistry
  • Succinates / metabolism
  • rab GTP-Binding Proteins / agonists*
  • rab GTP-Binding Proteins / chemistry
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Isoenzymes
  • Lipid Bilayers
  • Liposomes
  • MYO5B protein, human
  • N(alpha),N(alpha)-bis(carboxymethyl)-N(epsilon)-(1,2-dioleyl-sn-glycero-3-succinyl)-L-lysine
  • Oleic Acids
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Succinates
  • MYO5A protein, human
  • polyhistidine
  • Histidine
  • Guanosine Triphosphate
  • Myosin Type V
  • rab11 protein
  • Myosin Heavy Chains
  • rab GTP-Binding Proteins
  • Lysine