The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross‐linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross‐linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.
Various models for the transmembrane translocation of secreted and membrane-spanning proteins are reviewed in the light of available experimental data. Particular emphasis is placed on the crucial distinction between models where the protein passes through a hydrophilic 'tunnel' through the membrane, and models where translocation takes place through the non-polar intramembranous environment. It is concluded that rather simple reasoning in terms of a protein's behaviour in polar versus non-polar milieux apparently can tell us quite a lot about the process of translocation.
No abstract is provided for this article.
This chapter discusses the hydrophobicity concept and the various hydrophobicity scales that circulate in the literature. It deals with signal sequences and transmembrane segments, thus preparing the ground for a discussion of global models of protein export and membrane protein biogenesis. The concept of a hydrophobic effect, a tendency for nonpolar molecules or parts of molecules in aqueous solution to aggregate to reduce the nonpolar surface area exposed to water, has an immediate intuitive appeal, and it has been a central idea in many attempts to come to grips with the thermodynamics of protein structure. As most of the hydrophobicity scales in the literature agree in broad terms (be they empirical or statistical), most scales will yield similar results in any particular application. The central hydrophobic core is the most outstanding signature of a signal sequence. Proteins can bind to membranes in many ways. One useful distinction is between intrinsic and extrinsic membrane proteins, denoting, respectively, proteins spanning the nonpolar hydrocarbon interior of a membrane and proteins only associated with the (inner or outer) surface of the membrane. A number of characteristic features of start and stop signals are discussed in the chapter along with some interpretations of their possible functional relevance.
No abstract is provided for this article.
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.
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.
Tail‐anchored proteins are post‐translationally targeted and inserted into the endoplasmic reticulum membrane. They do not use the co‐translational signal‐recognition particle (SRP)‐dependent pathway, but rather utilize an ill‐defined, ATP‐dependent mechanism. Here, we show that a tail‐anchored protein can be cleaved by signal peptidase and that the sequence requirements for efficient cleavage seem to be the same as for cleavage of co‐translationally targeted SRP‐dependent proteins.
This volume of the Annual Review of Biochemistry contains three reviews on current developments in membrane protein research: Grigoryan et al. "Transmembrane Communication: General Principles and Lessons from the Structure and Function of the M2 Proton Channel, K⁺ Channels, and Integrin Receptors," Hagan et al. "β-Barrel Membrane Protein Assembly by the Bam Complex," and Dalbey et al. "Assembly of Bacterial Inner Membrane Proteins." In this short introduction, I discuss these reviews in the larger context of where the field of membrane protein biochemistry is heading.
No abstract is provided for this article.
Significance The orientations of multispanning membrane proteins in the membrane are initially determined when they are cotranslationally inserted into the membrane. However, it is not always clear whether these initial orientations may be dynamic after the insertion step or not. The present study shows that even in the case of small membrane proteins that have only four transmembrane helices and have little bias to be inserted in one or the other orientation, the initially inserted orientation remains stable and is not dynamic, at least with respect to the orientation of the entire protein. This finding suggests that membrane proteins become correctly oriented already during or immediately after synthesis, as they are inserted into the membrane.
My scientific career has taken me from chemistry, via theoretical physics and bioinformatics, to molecular biology and even structural biology. Along the way, serendipity led me to work on problems such as the identification of signal peptides that direct protein trafficking, membrane protein biogenesis, and cotranslational protein folding. I’ve had some great collaborations that came about because of a stray conversation or from following up on an interesting paper. And I’ve had the good fortune to be asked to sit on the Nobel Committee for Chemistry, where I am constantly reminded of the amazing pace and often intricate history of scientific discovery. Could I have planned this? No way! I just went with the flow … My scientific career has taken me from chemistry, via theoretical physics and bioinformatics, to molecular biology and even structural biology. Along the way, serendipity led me to work on problems such as the identification of signal peptides that direct protein trafficking, membrane protein biogenesis, and cotranslational protein folding. I’ve had some great collaborations that came about because of a stray conversation or from following up on an interesting paper. And I’ve had the good fortune to be asked to sit on the Nobel Committee for Chemistry, where I am constantly reminded of the amazing pace and often intricate history of scientific discovery. Could I have planned this? No way! I just went with the flow …
We have developed a method to reliably identify partial membrane protein topologies using the consensus of five topology prediction methods. When evaluated on a test set of experimentally characterized proteins, we find that approximately 90% of the partial consensus topologies are correctly predicted in membrane proteins from prokaryotic as well as eukaryotic organisms. Whole-genome analysis reveals that a reliable partial consensus topology can be predicted for approximately 70% of all membrane proteins in a typical bacterial genome and for approximately 55% of all membrane proteins in a typical eukaryotic genome. The average fraction of sequence length covered by a partial consensus topology is 44% for the prokaryotic proteins and 17% for the eukaryotic proteins in our test set, and similar numbers are found when the algorithm is applied to whole genomes. Reliably predicted partial topologies may simplify experimental determinations of membrane protein topology.
