Inhibition of Microtubule Assembly by HPC-1/Syntaxin 1A, An Exocytosis Relating Protein

Cell Structure and Function

Kimio Akagawa¹  Hirokazu Hotani²  Tomonori Fujiwara¹  Tadashi Shibuya²  Tomohiko J. Itoh² 

[1] Department of Physiology Kyorin University, School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-0004, Japan;Division of Biological Sciences, Graduate School of Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

关键词: tubulin;    HPC-1;    syntaxin 1A;    cross-linking;    dark-field microscopy;   

DOI  :  10.1247/csf.24.359

学科分类：分子生物学，细胞生物学和基因

来源: Japan Society for Cell Biology

【 摘 要 】

References(26)Cited-By(3)HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membrane protein, is believed to regulate the synaptic exocytosis as a component of t-SNARE. The distribution of the protein, however, is not restricted to the synaptic terminal, but it has been found to locate on the axonal membrane. When the expression of HPC-1 was suppressed, neurite sprouting was enhanced in cultured neurons. These findings suggest that HPC-1 possesses other functions than the regulation of the membrane fusion in neuro-transmitter release. Rather it may also participate in the morphogenesis of neurons through membrane fusion, and possibly through cytoskeleton. HPC-1 has a sequence resemble to the assembly promoting sequence of heat stable MAPs in residues 89-106, suggesting that it can bind tubulin and be involved in microtubule system. Thus, both the tubulin binding property and the effect on microtubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacking the C-terminal transmembrane region (HPC-ΔTM), which was overexpressed in E. coli. Affinity column chromatography showed that tubulin was found to bind HPC-1 directly. Synthetic peptide which corresponds to the residues 89-106 competitively inhibited the tubulin-HPC-1 binding, indicating that the sequence is responsible for the tubulin binding. In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubulin molecule. Light scattering measurement of microtubule polymerization showed that HPC-1 decreased the rate of the pure tubulin polymerization. Direct observation of single microtubules under dark-field microscopy showed that the growth rate of microtubule decreased by HPC-1. After shortening stopped, microtubules often spent attenuate phases, in which neither growing nor shortening was detected. When another mutant HPC-1 which is composed of residues 1-97 and lacks tubulin binding activity was used, however, the suppression of microtubule polymerization was not observed. These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which directly bind tubulin subunit and decrease the polymerization activity.

【 授权许可】

Unknown