Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles.
The Ral proteins are members of the Ras superfamily of GTPases. Because they reside in synaptic vesicles, we used transgenic mice expressing a dominant inhibitory form of Ral to investigate the role of Ral in neurosecretion. Using a synaptosomal secretion assay, we found that while K(+)-evoked secretion of glutamate was normal, protein kinase C-mediated enhancement of glutamate secretion was suppressed in the mutant mice. Since protein kinase C effects on secretion have been shown to be due to enhancement of the size of the readily releasable pool of synaptic vesicles docked at the plasma membrane, we directly measured the refilling of this readily releasable pool of synaptic vesicles after Ca(2+)-triggered exocytosis. Refilling of the readily releasable pool was suppressed in synaptosomes from mice expressing dominant inhibitory Ral. Moreover, we found that protein kinase C and calcium-induced phosphorylation of proteins thought to influence synaptic vesicle function, such as MARCKS, synapsin, and SNAP-25, were all reduced in synaptosomes from these transgenic mice. Concomitant with these studies, we searched for new functions of Ral by detecting proteins that specifically bind to it in cells. Consistent with the phenotype of the transgenic mice described above, we found that active but not inactive RalA binds to the Sec6/8 (exocyst) complex, whose yeast counterpart is essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. These findings demonstrate a role for Ral-GTPase signaling in the modulation of the readily releasable pool of synaptic vesicles and suggest the possible involvement of Ral-Sec6/8 (exocyst) binding in modulation of synaptic strength.
Polzin
Atsuko
A
Department of Biochemistry, Tufts University School of Medicine, Tufts-New England Medical Center, Boston, MA 02111, USA.
Shipitsin
Michail
M
Goi
Takanori
T
Feig
Larry A
LA
Turner
Timothy J
TJ
D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005089:Exocytosis / Q000187:drug effects; D005799:Genes, Dominant; D018698:Glutamic Acid / Q000032:analysis / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051379:Mice; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013574:Synaptosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins; D020725:ral GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; C490685:EXOC3 protein, human; C489607:EXOC4 protein, human; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C490683:Sec6l1 protein, mouse; C489608:Sec8l1 protein, mouse; D033921:Vesicular Transport Proteins; D015240:Phorbol 12,13-Dibutyrate; D018698:Glutamic Acid; D011493:Protein Kinase C; D020725:ral GTP-Binding Proteins; D002118:Calcium