An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST). Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop. The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent. It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage. The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements. Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies.
No abstract is provided for this article.
Like all cellular proteins, membrane proteins are synthesized by ribosomes. But unlike their soluble counterparts, highly hydrophobic membrane proteins require auxiliary machineries to prevent aggregation in aqueous cellular compartments. The principal machine is the translocon, which works in concert with ribosomes to manage the orderly insertion of alpha-helical membrane proteins directly into the endoplasmic reticulum membrane of eukaryotes or into the plasma membrane of bacteria. In the course of insertion, membrane proteins come into thermodynamic equilibrium with the lipid membrane, where physicochemical interactions determine the final three-dimensional structure. Much progress has been made during the past several years toward understanding the physical chemistry of membrane protein stability, the structure of the translocon machine, and the mechanisms by which the translocon selects and inserts transmembrane helices. We review this progress and consider the connection between the physical principles of membrane protein stability and translocon selection of transmembrane helices.
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.
No abstract is provided for this article.
No abstract is provided for this article.
In a current model of integration of multispanning membrane proteins into the endoplasmic reticulum, it is proposed that the transmembrane segments show alternating translocation initiation and stop-transfer functions. Here, we present evidence for a mode of cotranslational insertion in which an internal signal-anchor sequence with Nexo/Ccyt topology confers a transmembrane disposition onto a preceding hydrophilic segment, resulting in a topology where the hydrophilic segment apparently can slip back and forth across the membrane. Our results demonstrate that hydrophobicity is not, as hitherto thought, an absolute requirement for the formation of a transmembrane segment, and suggest that integral membrane proteins may contain hydrophilic transmembrane segments with a considerable freedom to move in relation to the membrane.
We have measured the minimum number of residues in a translocating polypeptide required to bridge the distance between the P-site in endoplasmic reticulum-bound ribosomes and the lumenally disposed active site of the oligosaccharyl transferase. The results suggest that a nascent chain may traverse the ribosome/translocase complex in a largely extended conformation, and that hydrophobic stop-transfer segments have a more compact, possibly α-helical conformation in the translocase. We have measured the minimum number of residues in a translocating polypeptide required to bridge the distance between the P-site in endoplasmic reticulum-bound ribosomes and the lumenally disposed active site of the oligosaccharyl transferase. The results suggest that a nascent chain may traverse the ribosome/translocase complex in a largely extended conformation, and that hydrophobic stop-transfer segments have a more compact, possibly α-helical conformation in the translocase.
No abstract is provided for this article.
Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.
In this study we have determined the position of the C-terminal end of the transmembrane domains of human integrin subunits (α2, α5, β1, β2) in microsomal membranes using the glycosylation mapping technique. In contrast to the common view, the transmembrane helices were found to extend roughly to Phe<sup>1129</sup> in α2, to Phe<sup>1026</sup> in α5, to Ile<sup>757</sup> in β1, and to His<sup>728</sup> in β2. The α-carbon of the conserved lysine present near the C-terminal end of the transmembrane helix (Lys<sup>1125</sup> in α2, Lys<sup>1022</sup> in α5, Lys<sup>752</sup> in β1, and Lys<sup>724</sup> in β2) is buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. A possible role for the conserved lysine in integrin function is discussed.
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 β isoforms expressed <i>in vitro</i> and in <i>Xenopus</i>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 <i>in vivo</i> 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.
Many integral membrane proteins use the same translocation machinery for membrane insertion as secretory proteins use to get across the membrane. This requires that transmembrane segments can be discriminated from other parts of the protein during membrane translocation, and further requires that the transmembrane segments can be moved laterally out of the translocation channel into the surrounding lipid. The molecular basis for this remarkable intramembraneous sorting event is a major focus of current studies of membrane protein biogenesis.
Determining gene functions from genomic sequences is a central goal of bioinformatics. Most purely computational approaches to this problem are based on the detection of genes with similar sequences. With the completion of fully sequenced genomes alternative approaches have become feasible. One such method is that of phylogenetic profiles. In this method a gene is described by its phylogenetic profile, i.e. a string that encodes the presence or absence of a homologous gene in other genomes. This string is then used to search for other genes with similar profiles. In this paper we briefly review the field as well as present an analysis on the performance of the method. We also discuss variations on this theme including inverse phylogenetic profiles and non-exact profiles using phylogenetic trees. In conclusion this indicates that phylogenetic profiles might be useful for some, but not all functional annotations. Functional annotation of genomes remains an important problem in genomics when no close ho mologs exist. Keywords: phylogenetic profile, phylgenetic reconstruction, gene prediction
Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic α‐helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C‐terminal region with the potential to form an amphiphilic β‐strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N‐tenninal domain similar to stroma‐targeting transit peptides attached to a C‐terminal apolar domain that share many characteristics with secretory signal peptides.
