Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.
Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.
复制标题
草履虫中参与吞噬体膜融合的蛋白质的克隆和测序。
DOI：
10.1091/mbc.10.4.1031
复制
发表时间：
1999
影响因子：
3.3
通讯作者：
Fok,AK
中科院分区：
文献类型：
--
作者：
Yamauchi,K;Aihara,MS;Ishida,M;Allen,RD;Fok,AK
用多微核草履虫（Parameciummultimicronucleatum）的C_5吞噬体抗原制备了单克隆抗体。为了确定其功能，克隆了编码C5的cDNA和基因组DNA。该抗原由315个氨基酸残基组成，预测分子量为36，594，与SDS-PAGE测定的值相似。序列比较发现与粟酒裂殖酵母蛋白和运动发酵单胞菌β-呋喃果糖苷酶蛋白C-末端的一半有很低但显著的同源性。C5蛋白的N端缺乏明显的跨膜结构域或信号序列，推测其为可溶性蛋白，而免疫荧光显示其存在于囊泡和消化泡的膜上。在最低程度透化但具有完整DV的细胞中，发现C5位于DV膜的胞质表面上。来自纯化的和KCl洗涤的DV的蛋白质的免疫印迹显示C5与DV膜紧密结合。冷冻电子显微镜还证实，C5是在细胞质表面的盘状囊泡，酸体，和溶酶体，细胞器与细胞咽膜融合，DV的阶段I（DV-I）和II（DV-II），分别。虽然C5更集中在成熟的比年轻的DV膜，引人注目的观察是，来自盘状囊泡的细胞咽膜几乎没有C5。大约80%的C5从盘状囊泡衍生的膜与细胞咽膜融合后失去。将mAb显微注射至C5极大地抑制了盘状囊泡与细胞咽膜的融合，从而抑制了盘状囊泡膜并入DV膜中。两者合计，这些结果表明，C5是一种膜蛋白，参与盘状囊泡与导致DV形成的细胞咽膜的结合和/或融合。
An mAb was raised to the C5 phagosomal antigen inParamecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with aSchizosaccharomyces pombeprotein and the C-terminal half of the β-fructofuranosidase protein ofZymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation.
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使用单克隆抗体区分草履虫吞噬体膜和阶段
DOI：
--
发表时间：
1986
期刊：
影响因子：
--
作者：
A. Fok;M. Ueno;R. Allen
通讯作者：
R. Allen
影响因子：
56.9
作者：
STURGILLKOSZYCKI, S;SCHLESINGER, PH;RUSSELL, DG
通讯作者：
RUSSELL, DG
消化系统膜：冻结裂纹证据，证明了甲虫的分化和流动。
影响因子：
7.8
作者：
Allen, R D;Staehelin, L A
通讯作者：
Staehelin, L A
尾草履虫吞噬溶酶体溶酶体膜和酸性磷酸酶的回收
影响因子：
7.8
作者：
R. Allen;A. Fok
通讯作者：
A. Fok
四膜虫和其他纤毛虫中的密码子使用。
DOI：
10.1111/j.1550-7408.1989.tb02679.x
发表时间：
1989
期刊：
The Journal of protozoology
影响因子：
--
作者：
Duane W. Martindale
通讯作者：
Duane W. Martindale