Lin Xian-Guang, Ming Min, Chen Mao-Rong, Niu Wei-Pin, Zhang Yong-Deng, Liu Bei, Jiu Ya-Ming, Yu Jun-Wei, Xu Tao, Wu Zheng-Xing
Key Laboratory of Molecular Biophysics, Ministry of Education, and Institute of Biophysics & Biochemistry, Huazhong University of Science & Technology, 430074 Wuhan, People's Republic of China.
Biochem Biophys Res Commun. 2010 Jul 2;397(3):526-31. doi: 10.1016/j.bbrc.2010.05.148. Epub 2010 May 31.
UNC-31 or its mammalian homologue, Ca(2+)-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca(2+)-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca(2+) level (pre-flash Ca(2+) was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca(2+) evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex.
UNC-31或其哺乳动物同源物——分泌型钙依赖性激活蛋白（CAPS），对于致密核心囊泡（DCV）和突触囊泡（SV）的胞吐作用必不可少。从N端到C端，UNC-31包含多个推定的功能结构域，包括动力蛋白激活蛋白1结合结构域（DBD）、C2结构域、PH结构域、（M）UNC-13同源结构域（MHD）和DCV结合结构域（DCVBD），本研究中我们检测了后四个结构域。我们利用UNC-31基因敲除的秀丽隐杆线虫来检测全长UNC-31与这四个结构域缺失突变体中的每一个异位表达能否挽救UNC-31的功能。全长UNC-31 cDNA挽救了秀丽隐杆线虫基因敲除突变体在ALA神经元中钙升高时的表型。令人惊讶的是，MHD缺失也部分挽救了UNC-31的胞吐功能，因为电容研究中使用的相对较高的钙水平（预闪光钙为450 nM）可以绕过MHD缺陷。尽管如此，其他三个结构域截短的cDNA对钙诱发的分泌几乎没有挽救作用。重要的是，这种基因敲除突变体挽救策略使得能够在从整个生物体到单个细胞的水平上进行生理学研究，如运动分析、神经肌肉接头处神经传递的药理学研究、体内神经肽释放测量以及囊泡对接分析。我们的结果表明，UNC-31的这些结构域各自支持UNC-31在囊泡胞吐作用中不同的连续分子作用，包括囊泡拴系和对接的步骤，这些步骤将囊泡与质膜连接起来，并随后通过启动可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）核心复合物的形成来引发囊泡。
