In mammalian cells, one-third of all polypeptides is transported into or through the ER-membrane via the Sec61-channel. While the Sec61-complex facilitates the transport of all polypeptides with amino-terminal signal peptides (SP) or SP-equivalent transmembrane helices (TMH), the translocating chain-associated membrane protein (now termed TRAM1) was proposed to support transport of a subset of precursors. To identify possible determinants of TRAM1 substrate specificity, we systematically identified TRAM1-dependent precursors by analyzing cellular protein abundance changes upon TRAM1 depletion in HeLa cells using quantitative label-free proteomics. In contrast to previous analysis after TRAP depletion, SP and TMH analysis of TRAM1 clients did not reveal any distinguishing features that could explain its putative substrate specificity. To further address the TRAM1 mechanism, live-cell calcium imaging was carried out after TRAM1 depletion in HeLa cells. In additional contrast to previous analysis after TRAP depletion, TRAM1 depletion did not affect calcium leakage from the ER. Thus, TRAM1 does not appear to act as SP- or TMH-receptor on the ER-membrane's cytosolic face and does not appear to affect the open probability of the Sec61-channel. It may rather play a supportive role in protein transport, such as making the phospholipid bilayer conducive for accepting SP and TMH in the vicinity of the lateral gate of the Sec61-channel.Abbreviations: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; RAMP, ribosome-associated membrane protein; SP, signal peptide; SR, SRP-receptor; SRP, signal recognition particle; TMH, signal peptide-equivalent transmembrane helix; TRAM, translocating chain-associated membrane protein; TRAP, translocon-associated protein.

中文翻译：

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TRAM1蛋白可能通过调节Sec61通道侧门附近的磷脂双层来支持ER蛋白的导入。

在哺乳动物细胞中，所有多肽的三分之一通过Sec61通道转运入或穿过ER膜。尽管Sec61复合物可促进带有氨基末端信号肽（SP）或SP等效跨膜螺旋（TMH）的所有多肽的运输，但提出了易位链相关膜蛋白（现称为TRAM1）来支持子集的运输的前体。为了确定TRAM1底物特异性的可能决定因素，我们通过使用无标记蛋白质组学分析HeLa细胞中TRAM1耗尽后细胞蛋白质丰度变化，系统地鉴定了TRAM1依赖性前体。与TRAP耗尽后的先前分析相反，对TRAM1客户的SP和TMH分析没有发现任何可解释其假定底物特异性的区别特征。为了进一步解决TRAM1机制，在HeLa细胞中TRAM1耗尽后进行了活细胞钙成像。与TRAP耗竭后的先前分析相比，TRAM1耗竭不影响ER内钙的泄漏。因此，TRAM1似乎不充当ER膜胞质面上的SP或TMH受体，并且似乎不影响Sec61通道的开放可能性。它可能在蛋白质运输中起支持作用，例如使磷脂双层有利于在Sec61通道侧门附近接受SP和TMH。OST，寡糖基转移酶；RAMP，核糖体相关膜蛋白；SP，信号肽；SR，SRP受体；SRP，信号识别粒子；TMH，相当于信号肽的跨膜螺旋；电车，易位链相关膜蛋白 TRAP，转运子相关蛋白。