Publications

Directionality in protein translocation across membranes: the N-tail phenomenon

Protein translocation normally starts from an N-terminal signal peptide and proceeds in an N-to-C-terminal direction. However, in certain integral membrane proteins an N-terminal tail is translocated even though it is not preceded by a signal peptide. In eukaryotic cells this process involves the normal Sec-machinery. In contrast, recent studies in Escherichia coli show that translocation of such N-terminal tails occurs by a mechanism that does not appear to involve the Sec proteins and is most efficient for short tails lacking positively charged residues. These novel observations suggest that the Sec-machinery has an inherent N-to-C-terminal directionality and cannot work ‘in reverse’.

Net N-C charge imbalance may be important for signal sequence function in bacteria

The net charge distribution in a region around the signal sequence cleavage site has been calculated for samples of 41 prokaryotic and 165 eukaryotic exported proteins. The results show that prokaryotic proteins in particular have a markedly higher incidence of acidic than of basic residues in this region. The possibility that a “dipolar” structure with a positive net charge difference between the N and C-terminal regions is important for signal sequence function in bacteria is suggested, and invoked to rationalize a number of known export-defective signal sequence mutations.

Probing Interplays between Human XBP1u Translational Arrest Peptide and 80S Ribosome

The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond 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 potencies. 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.

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths.

Research Article Summary

The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase.

Signal peptides (SPs) target proteins to the secretory pathway and are cleaved from the nascent chain once the translocase in the ER has been engaged. Signal-anchor (SA) sequences also interact transiently with the ER translocase, but are not cleaved and move laterally out of the translocase to become permanent membrane anchors. One obvious difference between SP and SA sequences is the considerably longer hydrophobic regions (h regions) of the latter. To study the interaction between SP/SA sequences and the ER translocase, we have constructed signal sequences with poly-Leu h regions ranging in length from 8 to 29 residues and have characterized their locations within the translocase using both a new assay that measures the minimum number of amino acids needed to span the distance between the COOH-terminal end of the h region and the active site of the oligosaccharyl transferase enzyme and an assay where the efficiency of signal peptidase catalyzed cleavage is measured. Our results suggest that SP and SA sequences are positioned differently in the ER translocase.

SignalP 4.0: discriminating signal peptides from transmembrane regions

The secretory signal peptide is a ubiquitous protein-sorting signal that targets its passenger protein for translocation across the endoplasmic reticulum membrane in eukaryotes and the cytoplasmic membrane in prokaryotes1. Many methods have been published for predicting signal peptides from the amino acid sequence, including SignalP2,3,4, PrediSi5, SPEPlip6, Signal-CF7, Signal-3L8 and Signal-BLAST9. A benchmark study done in 2009 found SignalP 3.0 to be the best method10. All these methods, however, have only limited ability to distinguish between signal peptides and N-terminal transmembrane helices. Both peptides are hydrophobic, but transmembrane helices typically have longer hydrophobic regions. Also, transmembrane helices do not have cleavage sites, but the cleavage-site pattern is in itself not sufficient to distinguish the two types of sequence. This is a substantial problem because a scan for signal peptides in any complete genome will yield a lot of false positive predictions from N-terminal transmembrane regions.

Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method

A new, simple method for predicting transmembrane segments in integral membrane proteins has been developed. It is based on low-stringency dot-plots of the query sequence against a collection of non-homologous membrane proteins using a previously derived scoring matrix [Cserzö et al., 1994, J. Mol. Biol., 243, 388-396]. This so-called dense alignment surface (DAS) method is shown to perform on par with earlier methods that require extra information in the form of multiple sequence alignments or the distribution of positively charged residues outside the transmembrane segments, and thus improves prediction abilities when only single-sequence information is available or for classes of membrane proteins that do not follow the 'positive inside' rule.

Introduction/Overview

This chapter introduces the gist of the complete book as well as the particular topic discussed in each chapter. All living cells contain proteins that carry out specialized functions within various subcellular membrane or aqueous spaces. Bacterial cells have at least one membrane that separates the inside of the cell from its environment. Most proteins are synthesized in the cytoplasm of the cell, except for a small number that are encoded in the mitochondrial and chloroplast genomes. This raises the question of how proteins are transported from the cytoplasm to other destinations within or outside of the cell. Proteins are imported directly from the cytoplasm into the endoplasmic reticulum (ER), mitochondria, peroxisomes, and chloroplasts by mechanisms that use a targeting sequence and a translocation machinery. In addition to importing proteins from the cytoplasm into the organelle, mitochondria and chloroplasts also export proteins from the mitochondrial matrix or the chloroplast stroma where proteins are encoded by their respective organellar genomes. Overall this chapter reveals that this book brings together a number of important topics in the protein localization field.

Improved Prediction of Signal Peptides: SignalP 3.0

We describe improvements of the currently most popular method for prediction of classically secreted proteins, SignalP. SignalP consists of two different predictors based on neural network and hidden Markov model algorithms, where both components have been updated. Motivated by the idea that the cleavage site position and the amino acid composition of the signal peptide are correlated, new features have been included as input to the neural network. This addition, combined with a thorough error-correction of a new data set, have improved the performance of the predictor significantly over SignalP version 2. In version 3, correctness of the cleavage site predictions has increased notably for all three organism groups, eukaryotes, Gram-negative and Gram-positive bacteria. The accuracy of cleavage site prediction has increased in the range 6–17% over the previous version, whereas the signal peptide discrimination improvement is mainly due to the elimination of false-positive predictions, as well as the introduction of a new discrimination score for the neural network. The new method has been benchmarked against other available methods. Predictions can be made at the publicly available web server http://www.cbs.dtu.dk/services/SignalP/

Cotranslational folding of a pentarepeat β-helix protein

It is becoming increasingly clear that many proteins start to fold cotranslationally, before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, i.e ., proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum – a homolog of the Fluoroquinolone Resistance Protein MfpA – using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling, and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo , and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.

Mitochondrial targeting sequences why ‘non‐amphiphilic’ peptides may still be amphiphilic

The notion that mitochondrial targeting peptides form amphiphilic α‐helices with one apolar and one polar, positively charged face is controversial, since some experimental results seem to imply that non‐amphiphilic targeting peptides can also function as import signals. However, the standard methods used to assess the amphiphilicity of a peptide may be misleading, since they do not take the flexibility of the amino acid side chains into account. To demonstrate this, we have developed a new method for calculating the amphiphilicity of helical peptides.

Murine astrotactins 1 and 2 have similar membrane topology and mature via endoproteolytic cleavage catalyzed by signal peptidase

Astrotactins 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking and synaptic plasticity in the brain, as well as in planar polarity pathways in skin. Here, we used glycosylation mapping and protease-protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes (RMs), and found that Astn2 has a cleaved N-terminal signal peptide (SP), an N-terminal domain located in the lumen of the RMs (topologically equivalent to the extracellular surface in cells), two transmembrane helices (TMHs), and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of SP cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second TMH; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase

Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Genome‐wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms

We have carried out detailed statistical analyses of integral membrane proteins of the helix‐bundle class from eubacterial, archaean, and eukaryotic organisms for which genome‐wide sequence data are available. Twenty to 30% of all ORFs are predicted to encode membrane proteins, with the larger genomes containing a higher fraction than the smaller ones. Although there is a general tendency that proteins with a smaller number of transmembrane segments are more prevalent than those with many, uni‐cellular organisms appear to prefer proteins with 6 and 12 transmembrane segments, whereas Caenorhabditis elegans and Homo sapiens have a slight preference for proteins with seven transmembrane segments. In all organisms, there is a tendency that membrane proteins either have manytransmembrane segments with short connecting loops or few transmembrane segments with large extra‐membraneous domains. Membrane proteins from all organisms studied, except possibly the archaeon Methanococcus jannaschii , follow the so‐called “positive‐inside” rule; i.e., they tend to have a higher frequency of positively charged residues in cytoplasmic than in extra‐cytoplasmic segments.

Emulating Membrane Protein Evolution by Rational Design

How do integral membrane proteins evolve in size and complexity? Using the small multidrug-resistance protein EmrE from Escherichia coli as a model, we experimentally demonstrated that the evolution of membrane proteins composed of two homologous but oppositely oriented domains can occur in a small number of steps: An original dual-topology protein evolves, through a gene-duplication event, to a heterodimer formed by two oppositely oriented monomers. This simple evolutionary pathway can explain the frequent occurrence of membrane proteins with an internal pseudo-two-fold symmetry axis in the plane of the membrane.

Prediction of transmembrane protein topology

Membrane proteins fulfil many critical cellular functions, and make up a large fraction of all proteins. In fact, it has recently been estimated that as many as 35–-40% of all yeast genes may code for integral membrane proteins (1,2). Yet, only a handful of high resolution membrane protein three-dimensional structures are known, making it even more important to extract as much structural information as possible from the amino acid sequences. This chapter will focus on the topology prediction problem: how to identify transmembrane segments and how to predict the overall orientation of the protein in the membrane.

A hidden Markov model for prediction transmembrane helices in proteinsequences

No abstract is provided for this article.