The membrane protein universe: what's out there and why bother?

Chances are that you have come across membrane proteins many times in your professional life: ion channels, aquaporins, G-protein-coupled receptors, drug resistance proteins. But it is also quite likely that you have never bothered to think about what the implications are of being a membrane protein, as opposed to a soluble protein. What is special about membrane proteins in terms of structure and function, how many membrane proteins are out there, how are they made in the cell? Welcome to the membrane protein universe!

Do protein-lipid interactions determine the recognition of transmembrane helices at the ER translocon?

Membrane-protein integration, folding and assembly processes in vivo depend on complex targeting, translocation, chaperoning, and sorting machineries that somehow read the 'molecular code' built into the nascent polypeptide, ultimately producing a properly folded protein integrated into the correct target membrane. Although the main molecular constituents and the basic mechanistic principles of many of these machines are known in outline, the codes remain poorly defined and there is little quantitative information on how protein sequence affects the final structure of membrane proteins. By carefully designing model protein constructs, we have derived the first true biological hydrophobicity scale and have been able to get a first impression of how the position of a given type of residue within a transmembrane segment affects its ability to promote membrane insertion.

Effect of acute hyperketonemia on the cerebral uptake of ketone bodies in nondiabetic subjects and IDDM patients.

Using R-beta-[1-(11)C]hydroxybutyrate and positron emission tomography, we studied the effect of acute hyperketonemia (range 0.7-1.7 micromol/ml) on cerebral ketone body utilization in six nondiabetic subjects and six insulin-dependent diabetes mellitus (IDDM) patients with average metabolic control (HbA(1c) = 8.1 +/- 1.7%). An infusion of unlabeled R-beta-hydroxybutyrate was started 1 h before the bolus injection of R-beta-[1-(11)C]hydroxybutyrate. The time course of the radioactivity in the brain was measured during 10 min. For both groups, the utilization rate of ketone bodies was found to increase nearly proportionally with the plasma concentration of ketone bodies (1.0 +/- 0.3 micromol/ml for nondiabetic subjects and 1.3 +/- 0.3 micromol/ml for IDDM patients). No transport of ketone bodies from the brain could be detected. This result, together with a recent study of the tissue concentration of R-beta-hydroxybutyrate in the brain by magnetic resonance spectroscopy, indicate that, also at acute hyperketonemia, the rate-limiting step for ketone body utilization is the transport into the brain. No significant difference in transport and utilization of ketone bodies could be detected between the nondiabetic subjects and the IDDM patients.

Prediction of organellar targeting signals.

The subcellular location of a protein is an important characteristic with functional implications, and hence the problem of predicting subcellular localization from the amino acid sequence has received a fair amount of attention from the bioinformatics community. This review attempts to summarize the present state of the art in the field.

Formation of helical hairpins during membrane protein integration into the endoplasmic reticulum membrane. Role of the N and C-terminal flanking regions.

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a common structural element in integral membrane proteins. Previous studies on the sequence determinants of helical hairpin formation have focussed on the role of polar and charged residues placed centrally in a long stretch of hydrophobic residues, and have yielded a "propensity scale" for the relative efficiency with which different residues promote the formation of helical hairpins. In this study, we shift our attention to the role of charged residues flanking the hydrophobic stretch. Clusters of charged residues are known to hinder membrane translocation, and thus flanking charged residues may conceivably force a long hydrophobic segment to form a helical hairpin even if there are no or only weakly turn-promoting residues in the hydrophobic stretch. We indeed find that Lys and, more surprisingly, Asp residues strongly affect helical hairpin formation when placed next to a poly-Leu-based transmembrane segment. We also find that a cluster of four consecutive Lys residues can affect the efficiency of helical hairpin formation even when placed approximately 30 residues downstream of the hydrophobic stretch. These observations have interesting implications for the way we picture membrane protein topogenesis within the context of the endoplasmic reticulum (ER) translocon.

Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli.

Escherichia coli is one of the most widely used vehicles to overexpress membrane proteins (MPs). Currently, it is not possible to predict if an overexpressed MP will end up in the cytoplasmic membrane or in inclusion bodies. Overexpression of MPs in the cytoplasmic membrane is strongly favoured to overexpression in inclusion bodies, since it is relatively easy to isolate MPs from membranes and usually impossible to isolate them from inclusion bodies. Here we show that green fluorescent protein (GFP), when fused to an overexpressed MP, can be used as an indicator to monitor membrane insertion versus inclusion body formation of overexpressed MPs in E. coli. Furthermore, we show that an overexpressed MP can be recovered from a MP-GFP fusion using a site specific protease. This makes GFP an excellent tool for large-scale MP target selection in structural genomics projects.

Inhibition of protein translocation across the endoplasmic reticulum membrane by sterols.

Cholesterol and related sterols are known to modulate the physical properties of biological membranes and can affect the activities of membrane-bound protein complexes. Here, we report that an early step in protein translocation across the endoplasmic reticulum (ER) membrane is reversibly inhibited by cholesterol levels significantly lower than those found in the plasma membrane. By UV-induced chemical cross-linking we further show that high cholesterol levels prevent cross-linking between ribosome-nascent chain complexes and components of the Sec61 translocon, but have no effect on cross-linking to the signal recognition particle. The inhibiting effect on translocation is different between different sterols. Our data suggest that the protein translocation machinery may be sensitive to changes in cholesterol levels in the ER membrane.

Effects of 'hydrophobic mismatch' on the location of transmembrane helices in the ER membrane.

We have studied the effects of 'hydrophobic mismatch' between a poly-Leu transmembrane helix (TMH) and the ER membrane using a glycosylation mapping approach. The simplest interpretation of our results is that the lumenal end of the TMH is located deeper in the membrane for both short (negative mismatch) and long (positive mismatch) TMHs than for poly-Leu segments of intermediate length. We further find that the position-specific effect of Lys residues on the location of short TMHs in the membrane varies with an apparent helical periodicity when the Lys residue is moved along the poly-Leu stretch. We discuss these findings in the context of models for peptide-lipid interactions during hydrophobic mismatch.

The internal repeats in the Na+/Ca2+ exchanger-related Escherichia coli protein YrbG have opposite membrane topologies.

We have determined the topology of the Escherichia coli inner membrane protein YrbG, a putative Na(+)/Ca(2+) exchanger with homology to a family of eukaryotic ion exchangers. Our results show that the two homologous halves of YrbG both have five transmembrane segments but opposite membrane orientations. This has implications for our understanding of the function of Na(+)/Ca(2+) exchangers and provides an example of "divergent" evolution of membrane protein topology.

Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.

We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C(in) topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/.

Consensus predictions of membrane protein topology.

We have explored the possibility that consensus predictions of membrane protein topology might provide a means to estimate the reliability of a predicted topology. Using five current topology prediction methods and a test set of 60 Escherichia coli inner membrane proteins with experimentally determined topologies, we find that prediction performance varies strongly with the number of methods that agree, and that the topology of nearly half of all E. coli inner membrane proteins can be predicted with high reliability (>90% correct predictions) by a simple majority-vote approach.

Charge pair interactions in a model transmembrane helix in the ER membrane.

We have examined the effects of Lys-Asp charge pair interactions on the position of a model poly-Leu transmembrane helix in the ER membrane using the so-called "glycosylation mapping" technique. Based on an analysis of a set of constructs containing pairs of positively charged Lys and negatively charged Asp residues in various positions in the model helix, we show that the helix is located deeper in the membrane when Lys and Asp are placed one helical turn apart than for other spacings of the two residues. These results suggest that salt-bridge formation between residues located on the same face of a transmembrane helix may reduce the free energy of membrane partitioning.

How proteins adapt to a membrane-water interface.

Membrane proteins present a hydrophobic surface to the surrounding lipid, whereas portions protruding into the aqueous milieu expose a polar surface. But how have proteins evolved to deal with the complex environment at the membrane-water interface? Some insights have been provided by high-resolution structures of membrane proteins, and recent studies of the role of individual amino acids in mediating protein-lipid contacts have shed further light on this issue. It now appears clear that the polar-aromatic residues Trp and Tyr have a specific affinity for a region near the lipid carbonyls, whereas positively charged residues extend into the lipid phosphate region.

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane.

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.

Predicting subcellular localization of proteins based on their N-terminal amino acid sequence.

A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.

Determinants of topogenesis and glycosylation of type II membrane proteins. Analysis of Na,K-ATPase beta 1 AND beta 3 subunits by glycosylation mapping.

The structural and molecular determinants that govern the correct membrane insertion and folding of membrane proteins are still ill-defined. By following the addition of sugar chains to engineered glycosylation sites (glycosylation mapping) in Na,K-ATPase beta isoforms expressed in vitro and in Xenopus oocytes, in combination with biochemical techniques, we have defined the C-terminal end of the transmembrane domain of these type II proteins. N-terminal truncation and the removal of a single charged residue at the N-terminal start of the putative transmembrane domain influence the proper positioning of the transmembrane domain in the membrane as reflected by a repositioning of the transmembrane domain, the exposure of a putative cryptic signal peptidase cleavage site, and the production of protein species unable to insert into the membrane. Glycosylation mapping in vivo revealed that the degree of glycosylation at acceptor sites located close to the membrane increases with the time proteins spend in the endoplasmic reticulum. Furthermore, core sugars added to such acceptor sites cannot be processed to fully glycosylated species even when the protein is transported to the cell surface. Thus, the glycosylation mapping strategy applied in intact cells is a useful tool for the study of determinants for the correct membrane insertion of type II and probably other membrane proteins, as well as for the processing of sugar chains in glycoproteins.

Glycosylation efficiency of Asn-Xaa-Thr sequons depends both on the distance from the C terminus and on the presence of a downstream transmembrane segment.

Statistical studies of N-glycosylated proteins have indicated that the frequency of nonglycosylated Asn-Xaa-(Thr/Ser) sequons increases toward the C terminus (Gavel, Y., and von Heijne, G. (1990) Protein Eng. 3, 433-442). Using in vitro transcription/translation of a truncated model protein in the presence of dog pancreas microsomes, we find that glycosylation efficiency of Asn-Xaa-Thr sequons indeed is reduced when the sequon is within approximately 60 residues of the C terminus. Surprisingly, the presence of a hydrophobic stop transfer sequence between the Asn-Xaa-Thr sequon and the C terminus results in a very different dependence of glycosylation efficiency on the distance to the C terminus, where the presence of the stop transfer segment inside the ribosome appears to cause a drastic drop in the level of glycosylation. We speculate that this may reflect a change in the structure of the ribosome/translocon complex induced by the stop transfer segment.

YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase.

In Escherichia coli, both secretory and inner membrane proteins initially are targeted to the core SecYEG inner membrane translocase. Previous work has also identified the peripherally associated SecA protein as well as the SecD, SecF and YajC inner membrane proteins as components of the translocase. Here, we use a cross-linking approach to show that hydrophilic portions of a co-translationally targeted inner membrane protein (FtsQ) are close to SecA and SecY, suggesting that insertion takes place at the SecA/Y interface. The hydrophobic FtsQ signal anchor sequence contacts both lipids and a novel 60 kDa translocase-associated component that we identify as YidC. YidC is homologous to Saccharomyces cerevisiae Oxa1p, which has been shown to function in a novel export pathway at the mitochondrial inner membrane. We propose that YidC is involved in the insertion of hydrophobic sequences into the lipid bilayer after initial recognition by the SecAYEG translocase.