Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells.
Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells.
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深入了解活上皮细胞中角蛋白中间丝的动态特性。
DOI：
10.1083/jcb.153.3.503
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Goldman,RD
中科院分区：
文献类型：
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作者：
Yoon,KH;Yoon,M;Moir,RD;Khuon,S;Flitney,FW;Goldman,RD
通过基质辅助激光解吸电离质谱结合数据库搜索分析纯化的COPII囊泡上所含的蛋白质。我们鉴定了四种已知的囊泡蛋白（Erv 14 p，Bet 1 p，Emp 24 p和Erv 25 p）和另外九种尚未定位于ER囊泡的蛋白（Yip 3 p，Rer 1 p，Erp 1 p，Erp 2 p，Erv 29 p，Yif 1 p，Erv 41 p，Erv 46 p和Emp 47 p）。使用抗体，我们证明，这些蛋白质被选择性和有效地包装到COPII囊泡。三个新鉴定的囊泡蛋白（Erv 29 p，Erv 41 p和Erv 46 p）代表未表征的跨物种保守的整合膜蛋白。进一步表征了Erv 41 p和Erv 46 p。这些蛋白共定位于ER和高尔基体膜，并存在于通过免疫沉淀分离的洗涤剂可溶性复合物中。缺乏Erv 41 p和/或Erv 46 p的酵母菌株是活的，但显示冷敏感性。Erv 41 p和Erv 46 p的表达水平是相互依赖的，因此在anerv 41 Δ菌株中Erv 46 p被降低，而在anerv 46 Δ菌株中未检测到Erv 41 p。当erv 41 Δ或erv 46 Δ等位基因与早期分泌途径中的其他突变相结合时，在一些双突变菌株中观察到生长表型的改变。一个无细胞的测定，再现ER和高尔基体之间的运输表明，删除Erv 41 p-Erv 46 p复合物的影响膜融合阶段的运输。
Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in anerv41Δ strain, and Erv41p was not detected in anerv46Δ strain. When theerv41Δ orev46Δ alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p–Erv46p complex influences the membrane fusion stage of transport.