COMPONENTS AND KINETICS IN EXOCYTOSIS

胞吐作用的组成部分和动力学

• 批准号: 6290227
• 负责人: JOSHUA ZIMMERBERG
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290227
• 关键词: calcium ion; cell membrane; egg /ovum; exocytosis; granule; membrane fusion; sea urchins

We have continued our combined biophysical and biochemical research on the molecular mechanisms of membrane fusion during exocytosis, the critical step in insulin, neurotransmitter, and immunoglobulin secretion, and in fertilization. First,expression library screening with a monoclonal antibody to mammalian SNAP-25 was used to identify a S.purpuratus SNAP-25 homologue. A single isoform of sea urchin ovary SNAP-25 was cloned. The clone encodes a 212 amino acid protein (Gen Bank accession no. AF061750). This SNAP-25 is highly conserved, throughout the whole amino acid sequence when aligned to SNAP-25 from several species. The recombinant protein has been expressed in E.coli as a fusion construct, purified, and used as a standard in quantitative evaluation of the amount of SNAP-25 in cortical vesicles. The preliminary data show that ~1000 copies of SNAP-25 are present per vesicle. Second, syntaxin and VAMP have been immunoprecipated from cortical vesicles and the peptide maps analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The syntaxin immunoprecipated and purified from CV had peptide compositions after digestion that matched those predicted from the cDNA for urchin ovary syntaxin. VAMP from CV was isolated and the tryptic peptides were examined. Although the peptide map of VAMP contained many of the peptides predicted from the cDNA sequence, a major mass peak was present that did not correspond to the peptides predicted for urchin ovary VAMP. A stage-specific urchin ovary cDNA library was rescreened for a VAMP clone which had an amino terminal peptide consistent with the masses seen with post source decay (PSD)fragmentation. Several positive clones were isolated which contained an amino terminal sequence consistent with that obtained from the isolated CV VAMP. The sequence of the sea urchin ovary VAMP was determined (Gen Bank accession no. AF151536). In addition, the cDNA library was screened for sequences of the amino terminal of VAMP corresponding to that predicted for the cDNA sequence reported in the database. No positive clones were obtained using that sequence, indicating that there is no urchin VAMP that matches the published amino terminal sequence. Further analysis of this tryptic peptide by PSDfragmentation was used to determine the amino acid sequence and composition. PSD analysis indicated that the peptide could be the amino terminal peptide, since the peptide contained an amino terminal N-acetyl alanine. Although the amino terminal sequence of the peptide was distinct from the cDNA clone, the C-terminal sequence corresponded to that predicated from the cDNA clone. This discrepancy between the published VAMP cDNA sequence and the amino acid sequence of the isolated VAMP points out the importance of confirming the structure of the expressed protein, and not relying only on cloning data for sequence determination. Third, the fusion of CV to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescence microscopy in combination with electrical recordings of membrane conductance. A strong binding of CV to protein-free planar membrane was observed in the absence of calcium. Calcium-induced fusion of CV was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy; and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a content-specific dye. The results indicate that CV have sufficient calcium-sensitive proteins and docking machinery for fusion to lipid membranes, and in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites access to new membranes.

我们继续结合生物物理学和生物化学研究膜融合的分子机制，在胞吐，胰岛素，神经递质和免疫球蛋白分泌的关键步骤，并在受精。首先，使用针对哺乳动物SNAP-25的单克隆抗体筛选表达文库以鉴定紫链霉菌SNAP-25同源物。克隆了海胆卵巢SNAP-25的单一同种型。该克隆编码212个氨基酸的蛋白质（Gen Bank登录号AF 061750）。当与来自几个物种的SNAP-25比对时，该SNAP-25在整个氨基酸序列中是高度保守的。重组蛋白已在大肠杆菌中表达为融合构建体，纯化，并用作皮质囊泡中SNAP-25量的定量评价标准。初步数据显示，每个囊泡存在约1000个SNAP-25拷贝。第二，突触融合蛋白和VAMP已免疫沉淀从皮质囊泡和肽图分析基质辅助激光解吸/电离（MALDI）质谱。从CV免疫沉淀和纯化的突触融合蛋白消化后的肽组合物匹配的预测从海胆卵巢突触融合蛋白的cDNA。从CV中分离VAMP并检查胰蛋白酶肽。虽然VAMP的肽图谱包含许多从cDNA序列预测的肽，但存在一个主要的质量峰，其不对应于海胆卵巢VAMP预测的肽。重新筛选阶段特异性海胆卵巢cDNA文库中的VAMP克隆，其氨基末端肽与源后衰变（PSD）片段化所见的质量一致。分离了几个阳性克隆，其含有与从分离的CV VAMP获得的序列一致的氨基末端序列。测定了海胆卵巢VAMP的序列（GenBank登录号AF 151536）。此外，筛选cDNA文库中与数据库中报道的cDNA序列预测序列相对应的VAMP氨基末端序列。使用该序列未获得阳性克隆，表明不存在与公开的氨基末端序列匹配的海胆VAMP。通过PSD片段化对该胰蛋白酶肽进行进一步分析，以确定氨基酸序列和组成。PSD分析表明该肽可能是氨基末端肽，因为该肽含有氨基末端N-乙酰基丙氨酸。虽然肽的氨基末端序列与cDNA克隆不同，但C-末端序列与从cDNA克隆预测的序列相对应。公开的VAMP cDNA序列和分离的VAMP的氨基酸序列之间的这种差异指出了确认表达的蛋白质的结构的重要性，而不仅仅依赖于用于序列测定的克隆数据。第三，CV平面磷脂双层膜的融合进行了研究，微分干涉对比度（DIC）和荧光显微镜结合膜电导的电记录。在不存在钙的情况下，观察到CV与无蛋白平面膜的强结合。钙诱导的CV融合检测使用两个独立的测定：损失的内容物的个别囊泡可见DIC显微镜;和囊泡内容物放电的平面膜检测的内容物特异性染料的荧光增加。结果表明，CV有足够的钙敏感蛋白和对接机制融合到脂膜，并在本地皮质颗粒融合位点是面向质膜。从质膜去除囊泡可以允许融合位点进入新的膜。