Inhibition of microtubule assembly by HPC-1/syntaxin 1A, an exocytosis relating protein

HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membr ane 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 membr ane. When the expression of HPC-1 was suppressed, neurite sprouting was enh anced in cultured neurons. These findings suggest that HPC-1 possesses othe r functions than the regulation of the membrane fusion in neurotransmitter release. Rather it may also participate in the morphogenesis of neurons thr ough membrane fusion, and possibly through cytoskeleton. HPC-1 has a sequen ce resemble to the assembly promoting sequence of heat stable MAPs in resid ues 89-106, suggesting that it can bind tubulin and be involved in microtub ule system. Thus, both the tubulin binding property and the effect on micro tubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacki ng the C-terminal transmembrane region (HPC-Delta TM), which was overexpres sed 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 compet itively inhibited the tubulin-HPC-l binding, indicating that the sequence i s responsible for the tubulin binding. In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubuli n molecule. Light scattering measurement of microtubule polymerization show ed 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 stoppe d, microtubules often spent attenuate phases, in which neither growing nor shortening was detected. When another mutant HPC-1 which is composed of res idues 1-97 and lacks tubulin binding activity was used, however, the suppre ssion of microtubule polymerization was not observed. These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which dire ctly bind tubulin subunit and decrease the polymerization activity.