Publications

Assembly of several inner membrane proteins—leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep—has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

The current best membrane-protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of membrane proteins. However, because the insertion of transmembrane helices into the membrane is the outcome of molecular interactions among protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of membrane-protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.

Xbp1, a protein involved in the unfolded protein response, is a rare example of a mammalian protein that contains a well-defined translational arrest peptide (AP). In order to define the critical residues in the Xbp1u AP, and to search for variants with stronger arrest potency than the wildtype Xbp1u AP, we have carried out a full mutagenesis scan where each residue in the AP was replaced by the other 19 natural amino acids. We find that 10 of the 21 mutagenized positions are optimal already in the wildtype Xbp1 AP, while certain mutations in the remaining residues lead to a strong increase in the arrest potency. Xbp1 has thus evolved to induce an intermediate level of translational arrest, and versions with much stronger arrest efficiency exist. We further show Xbp1-induced translational arrest is reduced in response to increased tension in the nascent chain, making it possible to carry out studies in mammalian systems of cotranslational processes such as membrane protein assembly and protein folding by using suitable Xbp1 AP variants as “force sensors”, as has been done previously in E. coli using bacterial APs.

A considerable fraction of life develops in the sea at temperatures lower than 15°C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lipid desaturases achieve both protection against oxygen and synthesis of lipids making the membrane fluid. A remarkable strategy for avoidance of reactive oxygen species generation is developed by P. haloplanktis , with elimination of the ubiquitous molybdopterin-dependent metabolism. The P. haloplanktis proteome reveals a concerted amino acid usage bias specific to psychrophiles, consistently appearing apt to accommodate asparagine, a residue prone to make proteins age. Adding to its originality, P. haloplanktis further differs from its marine counterparts with recruitment of a plasmid origin of replication for its second chromosome.

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.

Because of their hydrophobic and hydrophilic nature and the need for a lipid bilayer for retaining the native structure, membrane proteins are hard to study. Nevertheless, they are important, as many of our diseases are related to membrane proteins and around 60% of the different pharmaceutical drugs are directed against a membrane proteins [1]. There are many ways to study a protein, you can study function, structure, how the protein is targeted and inserted into its specific organelle, the interactions with other proteins or ligands etc. In the absence of a high-resolution structure, a topology model for a membrane protein is often useful. We have obtained reliable topologies for 546 of the membrane proteins going through the secretory pathways in S. cerevisiae by combining experimental data with topology prediction programs. In addition we have produced topology models for over 15,000 membrane proteins from 38 sequenced eukaryotic genomes using homology to the experimentally determined group. We also examined the growth rates and tolerance to certain stress conditions for our large set of clones that over-express membrane proteins. This provides important information both for structural studies of membrane proteins where large amounts of protein is needed for further studies, and for getting some insight in the function of specific proteins. Finally we have studied the integration of membrane proteins by the Tim23 translocon in the inner membrane of mitochondria. We have investigated the hydrophobicity required for efficient integration of transmembrane (TM) helices by Tim23. From this data we have derived an in vivo hydrophobicity scale for the insertion of different amino acids into the inner membrane of the mitochondria, and have made a comparison with a previously determined hydrophobicity scale for the ER translocon Sec61. We concluded that charged residues flanking the TM segment are of major importance for insertion into the membrane. We therefore further investigated the importance of charged residues flanking the first, weakly hydrophobic, TM segment in the mitochondrial inner membrane protein Mgm1p with regard to membrane insertion by the Tim23 complex.

We have characterized the cotranslational folding of two small protein domains of different folds—the α-helical N-terminal domain of HemK and the β-rich FLN5 filamin domain—by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (force-profile analysis, or FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, photoinduced electron transfer, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.