Tetraspanins are found in multicellular eukaryotes and are generally thought to act as scaffolding proteins, localizing multiple proteins to a specific region of the cell membrane. Activities for tetraspanins have been identified in several fundamental processes such as motility, cell adhesion, proliferation and viral entry. Tetraspanins are also key players in cancer development and progression. However, structural and biochemical information on tetraspanins is decidely limited, due in no small part to the difficulties associated with expressing eukaryotic membrane proteins. In this study, we have used GFP fusions of a library of human tetraspanin proteins to identify growth conditions for expression in Escherichia coli. Three tetraspanin-GFP proteins could be produced at high enough levels to allow subsequent purification, paving the way for future structural and biochemical studies.
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 Nin-Cin topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for Nout-Cin 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/.
Many proteins contain sorting signals that serve to target them for import into different subcellular compartments. Similar signals also guide the insertion of integral membrane proteins into lipid bilayers. This chapter describes current methods for detecting such signals in amino acid sequences. Many of the functional characteristics of proteins can be correlated with more or less well-defined linear motifs in their amino acid sequences. This chapter deals with motifs that are known to be important for intracellular protein sorting, i.e., with sequence elements that serve as “address labels” and allow the cell to transport a given class of proteins to a given subcellular organelle. As it turns out, related motifs also guide membrane proteins into the lipid bilayer. In most cases, these motifs are encoded within a simple, contiguous stretch of the chain, and are thus, quite easy to identify in the primary sequence. The understanding of the signals that guide the assembly of membrane proteins is sufficiently advanced to allow quite reliable predictions of topology from sequence, at least for bacterial inner membrane proteins.
Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression.
No abstract is provided for this article.
We describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non‐functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located ~30 residues downstream of the signal peptide. When this segment is deleted, normal SecA‐dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA‐independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper.
About the play: In modern drama, the agonizing nature of membrane protein work has not been adequately acknowledged. It is perhaps significant that the first attempt to bring this darker aspect of human existence into focus comes from a Scandinavian author, writing in the tradition of Ibsen and Strindberg but with a distinctly turn-of-the-millenium approach to the inner life of his characters: the despairing Dr K; the cynical Dr R with his post-modernistic life credo; the ambitious but unfeeling Dr C; the modern Übermensch, Dr B., with his almost Nietzschean view of human nature. This is a play that is brutally honest, yet full of empathy for the poor souls that get caught between the Scylla of unreachable scientific glory and the Charybdis of helpless mediocrity. James Glib-Burdock, drama critic for The Stratford Observer.
This article provides an overview of the use of mathematical and computer modelling in furthering the understanding of protein synthesis. In particular, we discuss issues such as the nature of the rate limiting step(s), error rates, tRNA-codon adaptation, codon bias, attenuation control, and problems of selection and error corrections, focussing on their theoretical treatment.
The topologies of α-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on anti-parallel homo-or hetero-dimeric Small Multidrug Resistance proteins, and examine whether the individual monomers can undergo reversible topological inversion (flip-flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane topology of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final topologies independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo .
