Vesicular Messages from Dental Biofilms for Neutrophils.

The encounter between dental biofilm and neutrophils in periodontitis remains elusive, although it apparently plays a crucial role in the periodontal pathology and constitutes a key topic of periodontology. Dental biofilm and neutrophils were isolated from orally healthy persons and patients with periodontitis. We investigated biofilm and its particle-shedding phenomenon with electron microscopy and nanoparticle tracking analysis (NTA); biofilm shedding-neutrophil interactions were examined ex vivo with epi-fluorescence microscopy. For this purpose, we used acellular dental biofilm shedding, purified lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) as activators, and the interleukin 8 receptor beta (CXCR2) inhibitor and the anti-interleukin 8 receptor alpha (CXCR1) antibody as modulators. The shedding of acellular dental biofilms overwhelmingly consists of bacterial extracellular vesicles (BEVs). The latter induced the moderate formation of neutrophil extracellular traps (NETs) in orally healthy subjects and a strong formation in patients with periodontitis. A CXCR2 inhibitor and an anti-CXCR1 antibody had a minor effect on NET formation. Neutrophils from patients with periodontitis exhibited NET hyper-responsiveness. BEVs were stronger inducers of NET formation than purified LPS and PMA. A plateau of neutrophil responsiveness is reached above the age of 40 years, indicating the abrupt switch of maladaptive trained immunity (TI) into the activated modus. Our results suggest that dental biofilms consist of and disseminate immense amounts of outer membrane vesicles (OMVs), which initiate NET formation via a non-canonical cytosolic LPS/caspase-4/11/Gasdermin D pathway. This modus of NET formation is independent of neutrophil elastase (NE), myeloperoxidase (MPO), peptidylarginine deiminase 4 (PAD4), and toll-like receptors (TLR). In periodontitis, the hyper-responsiveness of neutrophils to BEVs and the increased NET formation appear to be a consequence of TI.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER.

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.

Organization of the native ribosome-translocon complex at the mammalian endoplasmic reticulum membrane.

BACKGROUND: In eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex. SCOPE OF REVIEW: The aim of this review is to summarize the current structural knowledge about protein translocon components in mammals. MAJOR CONCLUSIONS: Various structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation. GENERAL SIGNIFICANCE: The protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases.

Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum.

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca(2+) leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca(2+) leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic ß-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca(2+) imaging to monitor the effects on ER Ca(2+) leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca(2+) leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca(2+) leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic ß-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease.

BiP-mediated closing of the Sec61 channel limits Ca2+ leakage from the ER.

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca(2+) leak channel and identified calmodulin as limiting Ca(2+) leakage in a Ca(2+)-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca(2+) imaging to monitor the effects of reduced levels of BiP on ER Ca(2+) leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca(2+) leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca(2+) leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca(2+) leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum.

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.

Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells.

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Characterization of a unique attachment organelle: Single-cell force spectroscopy of Giardia duodenalis trophozoites.

The unicellular parasite Giardia duodenalis is the causative agent of giardiasis, a gastrointestinal disease with global spread. In its trophozoite form, G. duodenalis can adhere to the human intestinal epithelium and a variety of other, artificial surfaces. Its attachment is facilitated by a unique microtubule-based attachment organelle, the so-called ventral disc. The mechanical function of the ventral disc, however, is still debated. Earlier studies postulated that a dynamic negative pressure under the ventral disc, generated by persistently beating flagella, mediates the attachment. Later studies suggested a suction model based on structural changes of the ventral discs, substrate clutching or grasping, or unspecific contact forces. In this study, we aim to contribute to the understanding of G. duodenalis attachment by investigating detachment characteristics and determining adhesion forces of single trophozoites on a smooth glass surface (RMS = 1.1 ± 0.2 nm) by fluidic force microscopy (FluidFM)-based single-cell force spectroscopy (SCFS). Briefly, viable adherent trophozoites were approached with a FluidFM micropipette, immobilized to the micropipette aperture by negative pressure, and detached from the surface by micropipette retraction while retract force curves were recorded. These force curves displayed novel and so far undescribed characteristics for a microorganism, namely, gradual force increase on the pulled trophozoite, with localization of adhesion force shortly before cell detachment length. Respective adhesion forces reached 7.7 ± 4.2 nN at 1 µm s-1 pulling speed. Importantly, this unique force pattern was different from that of other eukaryotic cells such as Candida albicans or oral keratinocytes, considered for comparison in this study. The latter both displayed a force pattern with force peaks of different values or force plateaus (for keratinocytes) indicative of breakage of molecular bonds of cell-anchored classes of adhesion molecules or membrane components. Furthermore, the attachment mode of G. duodenalis trophozoites was mechanically resilient to tensile forces, when the pulling speeds were raised up to 10 µm s-1 and adhesion forces increased to 28.7 ± 10.5 nN. Taken together, comparative SCSF revealed novel and unique retract force curve characteristics for attached G. duodenalis, suggesting a ligand-independent suction mechanism, that differ from those of other well described eukaryotes.

Influence of a hydroxyapatite suspension on 48-h dental biofilm formation in-situ.

OBJECTIVE: The aim of the present in-situ study was to investigate anti-adherent properties of mouthrinses containing hydroxyapatite (HAP) nanoparticles on oral biofilm formation. DESIGN: Biofilm was formed for 48 h on bovine enamel or dentine specimens that were fixed to maxillary splints and worn intraoral by six volunteers. During biofilm formation, rinsing was performed with sterile water, HAP (5%) or chlorhexidine (0.2%) according to two different rinsing protocols in order to assess substantivity. Scanning electron microscopy was used to investigate biofilm coverage of specimens, biofilm thickness and morphology. In addition, saliva samples were collected and analysed with transmission electron microscopy. RESULTS: Rinsing with sterile water or HAP resulted in 2.1 or 2.3 µm thick biofilms, respectively, covering more than half of specimen' surfaces. Despite single deposits of nanoparticles in saliva and biofilm, HAP did not inhibit biofilm formation. Chlorhexidine on the other hand significantly reduced biofilm thickness and coverage. CONCLUSIONS: Mouthrinses containing HAP nanoparticles showed no anti-adherent effects during 48 h of biofilm formation in-situ.

Is the proteomic composition of the salivary pellicle dependent on the substrate material?

PURPOSE: The use of dental restorative materials is a routine task in clinical dentistry. Upon exposure to the oral cavity, continuous adsorption of salivary proteins and other macromolecules to all surfaces occurs, representing the first step in dental biofilm formation. Different physico-chemical properties of substrate materials potentially influence the composition of the initial biofilm, termed pellicle. This study aimed at characterizing and comparing the individual proteomic composition of the 3-min pellicle formed on bovine enamel and six restorative materials. EXPERIMENTAL DESIGN: After chemical elution, pellicle proteins were identified by nano-LC-HR-MS/MS. Proteomic profiles were analyzed in terms of molecular weights, isoelectric points, molecular functions and compared to saliva to reveal substrate material-specific adsorption patterns. RESULTS: A total of 1348 different pellicle proteins were identified, with 187-686 proteins in individual 3-min pellicles. Unexpectedly, this yielded quite similar distribution patterns independent of the substrate materials. Furthermore, overall similar fold changes were obtained for the major part of commonly enriched or depleted proteins in the pellicles. CONCLUSIONS AND CLINICAL RELEVANCE: The current results point to a minor role of the substrate material on the proteomic composition of the 3-min pellicle and represent core data for understanding the complex surface interactions in the oral cavity.

Hydroxyapatite Pellets as Versatile Model Surfaces for Systematic Adhesion Studies on Enamel: A Force Spectroscopy Case Study.

Research into materials for medical application draws inspiration from naturally occurring or synthesized surfaces, just like many other research directions. For medical application of materials, particular attention has to be paid to biocompatibility, osseointegration, and bacterial adhesion behavior. To understand their properties and behavior, experimental studies with natural materials such as teeth are strongly required. The results, however, may be highly case-dependent because natural surfaces have the disadvantage of being subject to wide variations, for instance in their chemical composition, structure, morphology, roughness, and porosity. A synthetic surface which mimics enamel in its performance with respect to bacterial adhesion and biocompatibility would, therefore, facilitate systematic studies much better. In this study, we discuss the possibility of using hydroxyapatite (HAp) pellets to simulate the surfaces of teeth and show the possibility and limitations of using a model surface. We performed single-cell force spectroscopy with single Staphylococcus aureus cells to measure adhesion-related parameters such as adhesion force and rupture length of cell wall proteins binding to HAp and enamel. We also examine the influence of blood plasma and saliva on the adhesion properties of S. aureus. The results of these measurements are matched to water wettability, elemental composition of the samples, and the change in the macromolecules adsorbed over time on the surface. We found that the adhesion properties of S. aureus were similar on HAp and enamel samples under all conditions: Significant decreases in adhesion strength were found equally in the presence of saliva or blood plasma on both surfaces. We therefore conclude that HAp pellets are a good alternative for natural dental material. This is especially true when slight variations in the physicochemical properties of the natural materials may affect the experimental series.

Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting.

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.

BiP modulates the affinity of its co-chaperone ERj1 for ribosomes.

Ribosomes synthesizing secretory and membrane proteins are bound to the endoplasmic reticulum (ER) membrane and attach to ribosome-associated membrane proteins such as the Sec61 complex, which forms the protein-conducting channel in the membrane. The ER membrane-resident Hsp40 protein ERj1 was characterized as being able to recruit BiP to ribosomes in solution and to regulate protein synthesis in a BiP-dependent manner. Here, we show that ERj1 and Sec61 are associated with ribosomes at the ER of human cells and that the binding of ERj1 to ribosomes occurs with a binding constant in the picomolar range and is prevented by pretreatment of ribosomes with RNase. However, the affinity of ERj1 for ribosomes dramatically changes upon binding of BiP. This modulation by BiP may be responsible for the dual role of ERj1 at the ribosome, i.e. acting as a recruiting factor for BiP and regulating translation.

Silencing of the SEC62 gene inhibits migratory and invasive potential of various tumor cells.

Sec62 is part of the protein translocation apparatus in the membrane of the endoplasmic reticulum (ER). In yeast, Sec62 participates in the post-translational translocation of proteins into the ER, but its function in mammals remains elusive. Previously we described the amplification and over-expression of the SEC62 gene in prostate cancer cell lines and the protein has been described as a potential target gene in prostate cancer. In the current study we show that in the tumor tissue of prostate cancer patients Sec62 protein levels are elevated compared with tumor-free tissue derived from the same patients or from prostates of control group patients and that the higher Sec62 protein content correlates with an increasing de-differentiation of the cells. Therefore, up-regulation of Sec62 protein content indeed is a phenomenon associated with prostate cancer progression. Analysis of a multi-tissue tumor array showed that in addition to prostate cancer, overproduction of Sec62 is observed in various other tumors, most significantly in tumors of the lung and the thyroid. To examine the tumor-related functions of Sec62, we silenced the SEC62 gene in the prostate cancer cell-line PC3 as well as in a set of other tumor cell-lines with two different siRNAs. In general, after silencing of SEC62 the cell migration and the invasive potential of the cells was blocked or at least dramatically reduced while cell viability was hardly affected. Thus, the SEC62 gene may indeed be considered as a target gene in the therapy of various tumors.

Quantification of the Adhesion Strength of Candida albicans to Tooth Enamel.

Caries is one of the most prevalent diseases worldwide, which is caused by the degradation of the tooth enamel surface. In earlier research the opportunistic pathogen Candida albicans has been associated with the formation of caries in children. Colonization of teeth by C. albicans starts with the initial adhesion of individual yeast cells to the tooth enamel surface. In this study, we visualized the initial colonization of C. albicans yeast cells on pellicle-covered enamel by scanning electron microscopy. To quantitatively unravel the initial adhesion strength, we applied fluidic force microscopy-based single-cell force spectroscopy to examine the key adhesion parameters adhesion force, rupture length and de-adhesion work. We analyzed single saliva-treated or untreated yeast cells on tooth enamel specimens with or without salivary pellicle. Under all tested conditions, adhesion forces in the lower nanonewton range were determined. Furthermore, we have found that all adhesion parameters were enhanced on the pellicle-covered compared to the uncovered enamel. Our data suggest that initial adhesion occurs through a strong interaction between yeast cell wall-associated adhesins and the salivary pellicle. Future SCFS studies may show whether specific management of the salivary pellicle reduces the adhesion of C. albicans on teeth and thus contributes to caries prophylaxis.

ERj1p uses a universal ribosomal adaptor site to coordinate the 80S ribosome at the membrane.

Ribosomes translating secretory and membrane proteins are targeted to the endoplasmic reticulum membrane and attach to the protein-conducting channel and ribosome-associated membrane proteins (RAMPs). Recently, a new RAMP, ERj1p, has been identified that recruits BiP to ribosomes and regulates translational activity. Here we present the cryo-EM structure of a ribosome-ERj1p complex, revealing how ERj1p coordinates the ribosome at the membrane and how allosteric effects may mediate ERj1p's regulatory activity.

ERj1p has a basic role in protein biogenesis at the endoplasmic reticulum.

ERj1p is a membrane protein of the endoplasmic reticulum (ER) that can recruit the ER lumenal chaperone BiP to translating ribosomes. ERj1p can also modulate protein synthesis at initiation and is predicted to be a membrane-tethered transcription factor. Here we attribute the various functions of ERj1p to distinct regions within its cytosolic domain. A highly positively charged nonapeptide within this domain is necessary and sufficient for binding to ribosomes. Binding of ERj1p to ribosomes involves the 28S ribosomal RNA and occurs at the tunnel exit. Additionally, ERj1p has a dual regulatory role in gene expression: ERj1p inhibits translation in the absence of BiP, and another charged oligopeptide within the cytosolic domain of ERj1p mediates binding of the nuclear import factor importin beta and import into the nucleus, thereby paving the way for subsequent action on genomic DNA.

Deep Proteomic Insights into the Individual Short-Term Pellicle Formation on Enamel-An In Situ Pilot Study.

PURPOSE: Dental pellicle formation starts instantaneously after oral hygiene due to the adsorption of salivary proteins to all orally exposed surfaces. The pellicle acts as a physiological mediator, protects the tooth surface from mechanical damages and reduces acid-induced enamel demineralization. The aim of this pilot study is to identify and characterize individual proteomic profiles of the initial pellicle formed on dental enamel and to compare the profiles with the corresponding saliva to analyze specific adsorption patterns occurring during pellicle formation. EXPERIMENTAL DESIGN: The 3-min pellicle of five subjects formed in situ on bovine enamel is eluted chemically and analyzed separately by nano-mass spectrometry. The analysis of the corresponding saliva is conducted in parallel. RESULTS: Up to 498 pellicle proteins and up to 1032 salivary proteins are identified on an individual level. Comparison of the salivary and pellicle protein profiles demonstrates the pellicle formation to be highly individual. Nineteen proteins are significantly enriched in the 3-min pellicle of all subjects and 22 proteins are significantly depleted indicating that pellicle formation relies on selective adsorption. CONCLUSIONS AND CLINICAL RELEVANCE: The short-term enamel pellicle is composed of several hundreds of adsorbed salivary proteins and reveals a highly individual proteomic profile.

TRAM1 protein may support ER protein import by modulating the phospholipid bilayer near the lateral gate of the Sec61-channel.

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.