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. 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.