A Rab GTPase is required for homotypic assembly of the endoplasmic reticulum.
To define the requirements for the homotypic fusion of mammalian endoplasmic reticulum (ER) membranes, we have developed a quantitative in vitro enzyme-linked immunosorbent assay. This assay measures the formation of IgG (H2L2) following the fusion of ER microsomes containing either the heavy or light chain subunits. Guanine nucleotide dissociation inhibitor (GDI), a protein that extracts Rab GTPases in the GDP-bound form from membranes, potently inhibits fusion. Inhibition was not observed using GDI mutants defective in Rab binding. Kinetic analysis of the inhibitory effects of GDI revealed that Rab activation is required immediately preceding or coincident with fusion and that this step is preceded by a priming event requiring a member of the AAA ATPase family. Our results suggest that homotypic fusion of ER membranes requires Rab and that Rab activation is a transient event necessary for the formation of a fusion pore leading to the mixing of luminal contents of ER microsomes.
Turner
M D
MD
Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Plutner
H
H
Balch
W E
WE
D000818:Animals; D002474:Cell-Free System; D002645:Chickens; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D008861:Microsomes / Q000201:enzymology / Q000502:physiology; D011506:Proteins / Q000378:metabolism*
C086796:GDP dissociation inhibitor 1; D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D007074:Immunoglobulin G; D011506:Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins