A Functional Role for GTP-Binding Proteins in Synaptic Vesicle Cycling

Abstract

The squid giant synapse was used to test the hypothesis that guanosine-5'-triphosphate (GTP)-binding proteins regulate the local distribution of synaptic vesicles within nerve terminals. Presynaptic injection of the nonhydrolyzable GTP analog GTP gamma S irreversibly inhibited neurotransmitter release without changing either the size of the calcium signals produced by presynaptic action potentials or the number of synaptic vesicles docked at presynaptic active zones. Neurotransmitter release was also inhibited by injection of the nonhydrolyzable guanosine diphosphate (GDP) analog GDP beta S but not by injection of AIF4-. These results suggest that a small molecular weight GTP-binding protein directs the docking of synaptic vesicles that occurs before calcium-dependent neurotransmitter release. Depletion of undocked synaptic vesicles by GTP gamma S indicates that additional GTP-binding proteins function in the terminal at other steps responsible for synaptic vesicle replenishment.

A synthetic peptide of the N‐terminus of ADP‐ribosylation factor (ARF) inhibits regulated exocytosis in adrenal chromaffin cells

“…The specific inhibition of regulated exocytosis in chromaffin cells by hARF1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) implicates ARF as a key part of the exocytotic machinery. A small component of Ca*'-dependent exocytosis was uninhibited by the peptide.…”

“…Effect of hARF1(2-17) on GTPyS-and Ca*+-induced secretion. Cells were permeabilised for 10 min in buffer A containing 20pM digitonin in the presence or absence of 100,uM hARF1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and in the presence or absence of 100 PM GTP [S] prior to stimulation with Buffer A containing 300nM free Ca2', 300nM free Ca2+ plus 100 PM GTPyS or 10 pM free Ca2' in the presence or absence of 100 pM hARF1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Catecholamine released over a 20 min period was expressed as a percentage of total cellular catecholamine.…”

“…The rab3AL(3347) peptide was synthesised in the Department of Biochemistry, University of Liverpool. hARF1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), sec4 (1-15), cal(l-15) and SynA(217-231) peptides were synthesized by Multiple Peptide Systems (San Diego, CA, USA), character&d by mass spectrometry and purified by reverse-phase HPLC. All other chemicals were from Sigma (Poole, UK).…”

“…The N-terminus of ARF is essential for its function and a synthetic N-terminal ARF peptide inhibits ARF function, endoplasmic reticulum (ER)-to-Golgi transport, intra-Golgi transport and endocytic vesicle fusion [IO-I 31. We have examined the effect of synthetic hARFl (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and found that it inhibits Ca*'-and GTP~S-de~ndent secretion from permeabilised adrenal chroma~n cells whereas other peptides are ineffective. These results implicate ARF as a key component of the regulated exocytotic machinery and emphasise the similarity of the mechanisms involved in the vesicular fusion events throughout the secretory pathway.…”

Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

“…For the electron microscopic analysis, distances between the active zone plasma membrane and individual synaptic vesicles were measured, as described (24). Because active zones are typically spaced about 1 m apart, synaptic vesicles within a 500-nm radius surrounding the active zone were measured in order to ensure that each synaptic vesicle was counted only once.…”