No abstract is provided for this article.
This chapter discusses the ways in which the amino acid sequence of a membrane protein dictates its insertion into the membrane and the ways in which it determines the final three dimensional fold. The chapter also focuses on membrane protein assembly that is found in the inner membrane of the bacterium Escherichia coli (E. coli) and has led to the formulation of the “positive inside” rule. The challenge that must be addressed to understand the entire assembly process is whether the formation of the topology corresponds to a distinct step in the assembly pathway, or whether the formation of the topology and the formation of the entire 3D structure are inseparable processes. Periplasmic and outer membrane E. coli proteins normally make use of the so-called Sec machinery for translocation across the inner membrane.
Determining the subcellular localization of a protein is an important first step toward understanding its function. Here, we describe the properties of three well-known N-terminal sequence motifs directing proteins to the secretory pathway, mitochondria and chloroplasts, and sketch a brief history of methods to predict subcellular localization based on these sorting signals and other sequence properties. We then outline how to use a number of internet-accessible tools to arrive at a reliable subcellular localization prediction for eukaryotic and prokaryotic proteins. In particular, we provide detailed step-by-step instructions for the coupled use of the amino-acid sequence-based predictors TargetP, SignalP, ChloroP and TMHMM, which are all hosted at the Center for Biological Sequence Analysis, Technical University of Denmark. In addition, we describe and provide web references to other useful subcellular localization predictors. Finally, we discuss predictive performance measures in general and the performance of TargetP and SignalP in particular.
Proline residues are rarely found in the three most C‐terminal positions of bacterial signal peptides, and have never been found in position +1 immediately following the cleavage site. It was recently shown that a Pro +1 mutation in the E. coli maltose binding protein precursor not only prevents cleavage of the signal peptide but also inhibits the leader peptidase enzyme, resulting in cessation of cell growth (Barkocy‐Gallagher, G.A. and Bassford, P.J. (1992) J. Biol. Chem. (in press)). Since maltose binding protein is dependent on the sec machinery for translocation across the inner membrane, it was not clear if this ‘Pro +1 ’ effect was restricted to sec ‐dependent proteins, or whether it applies also to proteins that do not require the sec functions for translocation. We now present data suggesting that the striking phenotypic effects of Pro +1 mutations can be elicited also by sec ‐independent proteins.
. 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!
The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome–translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion.
No abstract is provided for this article.
No abstract is provided for this article.
The eukaryotic cell has a highly developed machinery for routing proteins to their correct intra- or extra-cellular locations. Short stretches of amino acids ("signal peptides") serve as address labels that are recognized by receptors on the surface of the appropriate organelle, where-upon translocation across one or more membranes ensues. Premature folding of the nascent protein is prevented by cytoplasmic "chaperones", i.e. proteins that bind to unfolded or partially folded nascent chains. After import into the organelle, other chaperones are required for catalyzing the final folding of the protein, and for promoting its correct assembly into oligomeric protein complexes. Transport along the secretory pathway (endoplasmic reticulum to Golgi to trans-Golgi to plasma membrane) is mediated by vesicles that bud from a donor compartment and fuse with an acceptor compartment.
This folder contains the raw data that was used for quantifications (and the quantifications in an Excel sheet). The raw data (.txt files called EasyQuant files) was extracted from autoradiographs of SDS-PAGE gels using the ImageGauge software from Fuji (associated with the Fuji gel scanner). This machine has since been discontinued (these data were collected 2014-2016). The .txt files were imported into EasyQuant and fitted to a Gaussian distribution automatically by the software (software developed in the Gunnar von Heijne lab by Dr. Rickard Hedman), and the Ffl (fraction full length) was calculated.
No abstract is provided for this article.
To study the sequence requirements for addition of O-linked N-acetylgalactosamine to proteins, amino acid distributions around 174 O-glycosylation sites were compared with distributions around non-glycosylated sites. In comparison with non-glycosylated serine and threonine residues, the most prominent feature in the vicinity of O-glycosylated sites is a significantly increased frequency of proline residues, especially at positions -1 and +3 relative to the glycosylated residues. Alanine, serine and threonine are also significantly increased. The high serine and threonine content of O-glycosylated regions is due to the presence of clusters of several closely spaced glycosylated hydroxy amino acids in many O-glycosylated proteins. Such clusters can be predicted from the primary sequence in some cases, but there is no apparent possibility of predicting isolated O-glycosylation sites from primary sequence data.
The sodium ion-dependent citrate carrier of <i>Klebsiella pneumoniae</i> (CitS) contains 12 hydrophobic potential transmembrane domains. Surprisingly, an alkaline phosphatase fusion study in <i>Escherichia coli</i> has suggested that only 9 of these domains are embedded in the membrane, and 3 are translocated to the periplasm (van Geest, M., and Lolkema, J. S. (1996) <i>J. Biol. Chem.</i>271, 25582–25589). To provide independent data on the topology and mode of membrane insertion of CitS, we have investigated its insertion into the endoplasmic reticulum (ER) membrane. By using <i>in vitro</i> translation of model proteins in the presence of dog pancreas microsomes, each of the putative transmembrane segments of CitS was assayed for its potency to insert into the ER membrane, both as an isolated segment as well as in the context of COOH-terminal truncation mutants. All 12 segments were able to insert into the membrane as N<sub>cyt</sub>-C<sub>lum</sub>signal anchor sequences. In a series of COOH-terminal truncation mutants, the segments inserted in a sequential way except for one segment, segment Vb, which was translocated to the lumen. Hydrophobic segments VIII and IX, which, according to the alkaline phosphatase fusion study, are in the periplasm of <i>E. coli</i>, form a helical hairpin in the ER membrane. These observations suggest a topology for CitS with 11 transmembrane segments and also demonstrate that the sequence requirements for signal anchor and stop transfer function are different.
