Publications

Large-scale protein overexpression phenotype screens provide an important complement to the more common gene knockout screens. Here, we have targeted the so far poorly understood Saccharomyces cerevisiae membrane proteome and report growth phenotypes for a strain collection overexpressing ≈600 C-terminally tagged integral membrane proteins grown both under normal and three different stress conditions. Although overexpression of most membrane proteins reduce the growth rate in synthetic defined medium, we identify a large number of proteins that, when overexpressed, confer specific resistance to various stress conditions. Our data suggest that regulation of glycosylphosphatidylinositol anchor biosynthesis and the Na + /K + homeostasis system constitute major downstream targets of the yeast PKA/RAS pathway and point to a possible connection between the early secretory pathway and the cells’ response to oxidative stress. We also have quantified the expression levels for >550 membrane proteins, facilitating the choice of well expressing proteins for future functional and structural studies.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.

Ever since the signal hypothesis was proposed in 1971, the exact nature of signal peptides has been a focus point of research. The prediction of signal peptides and protein subcellular location from amino acid sequences has been an important problem in bioinformatics since the dawn of this research field, involving many statistical and machine learning technologies. In this review, we provide a historical account of how position-weight matrices, artificial neural networks, hidden Markov models, support vector machines and, lately, deep learning techniques have been used in the attempts to predict where proteins go. Because the secretory pathway was the first one to be studied both experimentally and through bioinformatics, our main focus is on the historical development of prediction methods for signal peptides that target proteins for secretion; prediction methods to identify targeting signals for other cellular compartments are treated in less detail.

The ribosome stalling mechanism is a crucial biological process; yet its atomistic underpinning is still elusive. In this framework, the XBP1u translational arrest peptide (AP) plays a central role in regulating the Unfolded Protein Response (UPR) in eukaryotic cells. Here, we report multi-microseconds all atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potency. Enhanced sampling simulations allow investigating the AP release process of the different variants shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae .

α-helical integral membrane proteins critically depend on the correct insertion of their transmembrane α helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane α helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (Ncyt-Clum or Nlum-Ccyt) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the “missing hydrophobicity” in polytopic membrane proteins.

The TIM23 complex mediates membrane insertion of presequence‐containing mitochondrial proteins via a stop‐transfer mechanism. Stop‐transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop‐transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23‐mediated stop‐transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2‐deletion or ‐overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix‐facing positive charges, is reduced. These results imply that the Mgr2‐mediated gatekeeper function is important for controlling membrane sorting of marginal stop‐transfer signals.

No abstract is provided for this article.

Topogenic sequences direct the membrane topology of proteins by being recognized and decoded by integral membrane translocases. In this paper, we have compared the minimal sequence characteristics of helical-hairpin, reverse signal-anchor, and stop-transfer sequences in bacterial membrane proteins that use either the YidC or SecYEG translocases for membrane insertion. We find that a stretch composed of 3 leucines and 16 alanines is required for efficient membrane-anchoring of the M13 procoat protein that inserts by a helical hairpin mechanism, and that a stretch composed of only 19 alanines has a detectable membrane-anchoring ability. Similar results were obtained for the reverse signal-anchor sequence of the single-spanning Pf3 coat protein and for stop-transfer segments engineered into leader peptidase. We have also determined the contribution to the apparent free energy of membrane insertion of M13 procoat for all 20 amino acids. The relative order of the contributions is similar to that determined for a stop-transfer sequence in the mammalian endoplasmic reticulum, but the absolute difference between the contributions for the most hydrophobic and most hydrophilic residues is somewhat larger in the E. coli system. These results are significant because they define the features of a membrane protein transmembrane segment that induce lateral release from the YidC and Sec translocases into the lipid bilayer in bacteria.

No abstract is provided for this article.

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N‐terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild‐type protein, the N‐terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N‐terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N‐tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N‐terminal tail causes TM1 to adopt an orientation with its N‐terminal end in the cytoplasm. We suggest that the topology of the TM1‐TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology.

In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.