MEMBRANE TRANSPORT AND FUSION

膜运输和融合

• 批准号: 3778622
• 负责人: J 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/3778622
• 关键词: Baculoviridae; Insecta; alternatives to animals in research; biological transport; cell fusion; cell membrane; chimeric proteins; exocytosis; guanosine triphosphate; laboratory rat; liver; membrane fusion; membrane permeability; membrane proteins; microsomes; oxidation reduction reaction; protein structure; tissue /cell culture

Cells must merge membranes in order to mix aqueous spaces. We have made progress this year in developing new methods for the characterization of fusion pores, and the identification and characterization of eukaryotic fusion proteins. Using double whole-cell recording technique. junctional conductance was measured during baculovirus-mediated syncytia formation of insect cells. Fusion was triggered by low pH. Symmetrical currents in each cell of the pair measured the fusion pore conductance directly. It started as abrupt (0.1-0.2 ms rise-time) pore formation with a distribution of conductances centered at l nS.- widened slowly in the time scale of tens of seconds and then increased abruptly. Our previous method, time-resolved admittance measurement, was performed to compare two methods: both gave similar results. Thus our previous methods of measuring fusion pores was confirmed. The new method will allow us to improve the time resolution of the fusion pore conductance by 1-2 orders of magnitude. A multiple- frequency, digital lock-in amplifier was implemented on a microcomputer to extend our measurements of fusion pores so as to calculate the fusion pore conductance more precisely in the presence of noise. To determine the proteins involved in fusion pore formation, thiol group sensitivity was studied. Amino-dextran derivatives of the reversible thiol reagent SPDP inhibit exocytosis inversely with size. This implies that the susceptible thiol groups are not buried deeply but are in a relatively exposed hydrophilic environment. Thiol-reactive proteins are involved in triggering fusion rather than downstream events, since no fusion is seen upon the removal of the SPDP block by DTT treatment. GTP-triggered fusion of rat liver microsomes was strongly inhibited by sodium metaperiodate. The restorative effect of DTT indicates that at least the reversible portion of the inhibition is caused by oxidation of protein vicinal thiol groups with the formation of disulfide bonds. Selective labeling of periodate-sensitive thiols revealed one major labeled polypeptide with M.W. of 15 kDa. The correlation between oxidation and reduction of the protein and inhibition and restoration of the microsomal fusion, respectively, suggests that this protein may be part of the GTP-dependent microsomal fusion machinery.

细胞必须合并膜以混合水空间。 我们有 今年在开发新方法方面取得了进展， 融合孔的表征，以及鉴定和 真核融合蛋白的表征。 采用双 全细胞记录技术测量结电导 在杆状病毒介导的昆虫细胞的合胞体形成期间。 融合由低pH触发。每个细胞中的对称电流 两人直接测量了融合孔电导。 它 开始为突然（0.1-0.2 ms上升时间）孔形成， 电导的分布以lnS为中心。慢慢扩大， 几十秒的时间尺度，然后突然增加。 我们 以前的方法，时间分辨导纳测量， 进行比较两种方法：两者都给出了类似的结果。 因此 证实了我们先前测量融合孔的方法。 的 新的方法将使我们能够提高时间分辨率的 融合孔电导提高1-2个数量级。 一个多重的- 频率，数字锁定放大器上实现的 微型计算机扩展我们的测量融合孔，以便 更精确地计算融合孔电导， 噪音的存在。 为了确定融合过程中涉及的蛋白质 孔形成，巯基敏感性进行了研究。氨基葡聚糖 可逆巯基试剂SPDP的衍生物抑制胞吐作用 与尺寸成反比。这意味着敏感的巯基 不是埋得很深，而是相对暴露的亲水性 环境 巯基反应蛋白参与触发 融合，而不是下游事件，因为没有融合被视为 DTT治疗解除SPDP阻滞。 GTP触发 钠离子对大鼠肝微粒体的融合有强烈的抑制作用 偏高碘酸盐。 DTT的修复效果表明， 至少抑制的可逆部分是由以下原因引起的： 蛋白质邻位硫醇基团的氧化， 二硫键 高碘酸盐敏感硫醇的选择性标记 发现一个主要的标记多肽，M.W. 15 kDa。的 蛋白质的氧化和还原之间的相关性， 分别抑制和恢复微粒体融合， 这表明这种蛋白质可能是GTP依赖的 微粒体融合机制