GTP CLEAVAGE BY THE SMALL GTP-BINDING PROTEIN RAB3A IS ASSOCIATED WITH EXOCYTOSIS OF SYNAPTIC VESICLES INDUCED BY ALPHA-LATROTOXIN

STAHL B, Fischer von Mollard G, WALCHSOLIMENA C, JAHN R. GTP CLEAVAGE BY THE SMALL GTP-BINDING PROTEIN RAB3A IS ASSOCIATED WITH EXOCYTOSIS OF SYNAPTIC VESICLES INDUCED BY ALPHA-LATROTOXIN. JOURNAL OF BIOLOGICAL CHEMISTRY. 1994;269(40):24770-24776.Neurotransmitter release from presynaptic nerve terminals is a highly regulated form of exocytosis. Small GTP-binding proteins of the Rab family have been proposed to act as central regulators in this process that cycle between a GTP- and GDP-bound form. Previous work has shown that the synaptic vesicle protein Rab3A undergoes a membrane association-dissociation cycle that is associated with neurotransmitter release. Using isolated nerve terminals as our model system, we have now analyzed the GDP/GTP status of Rab3A. Synaptic vesicle-bound Rab3A was almost exclusively in the GTP form whereas cytosolic Rab3A contained only GDP. Approximately equal amounts of GTP and GDP were found in the pool of Rab3A localized to a membrane fraction containing plasma membrane-synaptic vesicle complexes. In contrast to Rab3A, Rab5 (an endosomal G-protein) was predominantly GDP-bound in all analyzed compartments. To analyze whether Rab3A-bound GTP is cleaved during exocytosis, synaptosomes were stimulated with alpha-latrotoxin, the active component of black widow spider venom. This resulted in massive exocytosis. A significant increase of the GDP/GTP ratio of Rab3A was observed under these conditions that was not due to a nonspecific loss of high energy nucleotides. Our findings suggest that cleavage of Rab3A-bound GTP is a crucial step in regulated exocytosis of synaptic vesicles

LOCALIZATION OF RAB5 TO SYNAPTIC VESICLES IDENTIFIES ENDOSOMAL INTERMEDIATE IN SYNAPTIC VESICLE RECYCLING PATHWAY

Fischer von Mollard G, STAHL B, WALCHSOLIMENA C, et al. LOCALIZATION OF RAB5 TO SYNAPTIC VESICLES IDENTIFIES ENDOSOMAL INTERMEDIATE IN SYNAPTIC VESICLE RECYCLING PATHWAY. EUROPEAN JOURNAL OF CELL BIOLOGY. 1994;65(2):319-326.After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain. Rab5 is a major synaptic vesicle protein. Immunoisola tion of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor mediated endocytosis pathway