Hiromichi Shirataki

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Rab3A small GTP‐binding protein in Ca2+‐dependent exocytosis

There exists a small GTP‐binding protein (G protein) superfamily, consisting of more than 50 members, from yeast to mammal. The Rab family belongs to this superfamily and is implicated in intracellular vesicle trafficking. Rab3A small G protein is a member of the Rab3 subfamily which belongs to this Rab family. The regulators and downstream targets of Rab3A have been isolated, and evidence is accumulating that Rab3A and these Rab3A‐interacting proteins are involved in Ca2+‐dependent exocytosis, particularly in neurotransmitter release from nerve terminals.

Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine- and Ca2+-dependent manner

Rabphilin-3A is a putative target protein for Rab3A/Smg 25A, which is a member of the Ras-related small GTP-binding protein and implicated in neurotransmitter release from the synapse. Rabphilin-3A is composed of two functionally different domains: the N-terminal Rab3A-binding and the C-terminal phosphatidylserine- and Ca(2+)-binding domains. The C-terminal domain has two copies of an internal repeat that are homologous to the C2 domains of protein kinase C, synaptotagmin, and phospholipase A2 and C-gamma 1, which are known to bind phosphatidylserine and Ca2+. In this study, we attempted to identify the Rabphilin-3A-interacting molecule in bovine brain by use of an overlay assay technique. The 32P-labeled C-terminal fragment of Rabphilin-3A (281-704 amino acids) bound to a protein molecule with a M(r) of about 115 kDa which was immobilized on a nitrocellulose sheet. This protein was highly purified and characterized. The binding of the 32P-labeled C-terminal fragment to this protein was dependent on both phosphatidylserine and Ca2+, and inhibited by an excess amount of the C-terminal fragment and the C2 domain fragment (396-704 amino acids) but not by the N-terminal fragment (1-280 amino acids). These results indicate that Rabphilin-3A binds to a protein molecule with a M(r) of 115 kDa through the C2 domain in the presence of phosphatidylserine and Ca2+.

Rabphilin-3: a target molecule for Rab3 small G proteins.

This chapter describes the purification method for HA-tagged Rabphilin- 3 from the membrane fraction of overexpressing Spodoptera frugiperda cells (Sf9 cells). The chapter then describes the methods for detecting the interactions of Rabphilin-3 with α-actinin and rabaptin5, and the effects of Rabphilin-3 on the α-actinin-induced actin filament bundling and the receptor-mediated endocytosis. The steps used in the purification of HA-tagged Rabphilin-3 are as follows: (1) preparation of the membrane fraction from Sf9 cells, (2) heparin-Sepharose CL-6B column chromatography, and (3) Superose 12 HR10/30 column chromatography. The HA-tagged N-terminal fragment (amino acids 1–280) and HAtagged C-terminal fragment (amino acids 396–704) of Rabphilin-3 are purified by the same procedures as described above except that the Sf9 cells expressing each fragment are used. It has been shown that abnormalities of synaptic transmission and synaptic plasticity, which are observed in Rab3A-deficient mice, are not observed in Rabphilin-3-deficient mice. However, this does not necessarily indicate that Rabphilin-3 is not involved in neurotransmitter release. It is possible that Rim, another target molecule for Rab3A, compensates for the loss of function of Rabphilin-3 in the mice that abnormalities of synaptic transmission and synaptic plasticity, which are still not detected by the methods performed.