To elucidate the redundancy in the components for the targeting of membrane proteins to the endoplasmic reticulum (ER) and/or their insertion into the ER membrane under physiological conditions, we previously analyzed different human cells by label-free quantitative mass spectrometry. The HeLa and HEK293 cells had been depleted of a certain component by siRNA or CRISPR/Cas9 treatment or were deficient patient fibroblasts and compared to the respective control cells by differential protein abundance analysis. In addition to clients of the SRP and Sec61 complex, we identified membrane protein clients of components of the TRC/GET, SND, and PEX3 pathways for ER targeting, and Sec62, Sec63, TRAM1, and TRAP as putative auxiliary components of the Sec61 complex. Here, a comprehensive evaluation of these previously described differential protein abundance analyses, as well as similar analyses on the Sec61-co-operating EMC and the characteristics of the topogenic sequences of the various membrane protein clients, i.e., the client spectra of the components, are reported. As expected, the analysis characterized membrane protein precursors with cleavable amino-terminal signal peptides or amino-terminal transmembrane helices as predominant clients of SRP, as well as the Sec61 complex, while precursors with more central or even carboxy-terminal ones were found to dominate the client spectra of the SND and TRC/GET pathways for membrane targeting. For membrane protein insertion, the auxiliary Sec61 channel components indeed share the client spectra of the Sec61 complex to a large extent. However, we also detected some unexpected differences, particularly related to EMC, TRAP, and TRAM1. The possible mechanistic implications for membrane protein biogenesis at the human ER are discussed and can be expected to eventually advance our understanding of the mechanisms that are involved in the so-called Sec61-channelopathies, resulting from deficient ER protein import.
Previous electrophysiological experiments characterized the Sec61 complex, which provides the aqueous path for entry of newly‐synthesized polypeptides into the mammalian endoplasmic reticulum, as a highly dynamic channel that, once activated by precursor proteins, fluctuates between main open states with mean conductances of 220 and 550 pS. Millimolar concentrations of lanthanum ions simultaneously restricted the dynamics of the Sec61 channel and inhibited translocation of polypeptides. Molecular modeling indicates that lanthanum binding sites cluster at the putative lateral gate of the Sec61 complex and suggests that structural flexibility of the lateral gate is essential for channel and protein transport activities of the Sec61 complex.
The folding kinetics of two luciferases were studied after synthesis in reticulocyte lysates to investigate whether molecular chaperones and/or folding catalysts are involved in the folding reactions. Two bacterial luciferases were used as model proteins: heterodimeric Vibrio harveyi luciferase (LuxAB), and a monomeric luciferase fusion protein (Fab2). Data indicate that folding of these enzymes to the native state occurs in the translation system, and that the extent of folding can be quantified. It was found that (i) folding of LuxAB and Fab2 can clearly be separated in time from synthesis, (ii) folding of Fab2 and LuxAB is slow because it involves either transient (Fab2) or permanent (LuxAB) interaction of polypeptides, (iii) preservation of the assembly competent state of LuxA and/or LuxB and folding of Fab2 depend on ATP-hydrolysis, (iv) folding of Fab2 and LuxAB is partially sensitive to cyclosporin A (CsA) and FK506, i.e. inhibitors of two distinct peptidylprolyl cis/trans-isomerases. Thus, bacterial luciferases provide a unique system for direct measurement of the effects of ATP-dependent molecular chaperones on protein folding and enzyme assembly in reticulocyte lysates. Furthermore, these two luciferases provide the first direct evidence documenting the involvement of peptidylprolyl cis/trans-isomerases in protein biogenesis in a eukaryotic cytosol. The folding kinetics of two luciferases were studied after synthesis in reticulocyte lysates to investigate whether molecular chaperones and/or folding catalysts are involved in the folding reactions. Two bacterial luciferases were used as model proteins: heterodimeric Vibrio harveyi luciferase (LuxAB), and a monomeric luciferase fusion protein (Fab2). Data indicate that folding of these enzymes to the native state occurs in the translation system, and that the extent of folding can be quantified. It was found that (i) folding of LuxAB and Fab2 can clearly be separated in time from synthesis, (ii) folding of Fab2 and LuxAB is slow because it involves either transient (Fab2) or permanent (LuxAB) interaction of polypeptides, (iii) preservation of the assembly competent state of LuxA and/or LuxB and folding of Fab2 depend on ATP-hydrolysis, (iv) folding of Fab2 and LuxAB is partially sensitive to cyclosporin A (CsA) and FK506, i.e. inhibitors of two distinct peptidylprolyl cis/trans-isomerases. Thus, bacterial luciferases provide a unique system for direct measurement of the effects of ATP-dependent molecular chaperones on protein folding and enzyme assembly in reticulocyte lysates. Furthermore, these two luciferases provide the first direct evidence documenting the involvement of peptidylprolyl cis/trans-isomerases in protein biogenesis in a eukaryotic cytosol.
Importing proteins into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves the targeting of precursor proteins to the ER and their insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining the depletion of the central hSnd2 component from HeLa cells with proteomic and differential protein abundance analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals.
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.
