Assembly of membrane-bound respiratory complexes by the Tat protein-transport system

The Tat protein-export system serves to translocate folded proteins, often containing redox cofactors, across the bacterial inner membrane. Substrate proteins are directed to the Tat apparatus by distinctive N-terminal signal peptides containing a consensus SRRxFLK 'twin-arginine' motif. Here we review recent studies of the Tat system with particular emphasis on the assembly of membrane-bound respiratory complexes. We discuss the connection between Tat targeting and topological organisation of the complexes and consider the role of chaperone proteins in cofactor insertion and Tat targeting. The crystal structure of Escherichia coli formate dehydrogenase-N demonstrates that some Tat substrates are integral membrane proteins. Sequence analysis suggests that one-quarter of all traffic on the E. coli Tat pathway is inner-membrane proteins.     

Assembly and overexpression of membrane proteins in Escherichia coli

The bacterium Escherichia coli is one of the most popular model systems to study the assembly of membrane proteins of the so-called helix-bundle class. Here, based on this system, we review and discuss what is currently known about the assembly of these membrane proteins. In addition, we will briefly review and discuss how E. coli has been used as a vehicle for the overexpression of membrane proteins.     

On the targeting and membrane assembly of the Escherichia coli outer membrane porin, PhoE

Abstract Within gram-negative bacteria such as Escherichia coli , the outer membrane porins provide a relatively non-specific uptake route which is utilised by a wide range of solutes including many antibiotics. Understanding the targeting and membrane assembly of these proteins is therefore of importance and this mini review aims to discuss this process in light of present knowledge.     

Mechanisms of YidC-mediated insertion and assembly of multimeric membrane protein complexes

The YidC protein fulfills a dual and essential role in the assembly of inner membrane proteins in Escherichia coli. Besides interacting with transmembrane segments of newly synthesized membrane proteins that insert into the membrane via the SecYEG complex, YidC also functions as an independent membrane protein insertase and assists in membrane protein folding. Here, we discuss the mechanisms of YidC substrate recognition and membrane insertion with emphasis on its role in the assembly of multimeric membrane protein complexes such as the F1F0-ATP synthase.     

Biomolecular condensates in epithelial junctions

Epithelial junctions are transmembrane protein complexes that regulate cell adhesion, cell polarity, tissue permeability, and tissue mechanics. Most junctional complexes contain membrane attached cytoplasmic plaques that regulate junction assembly and are composed of multivalent scaffold proteins. In this review, we discuss phase separation of multivalent proteins as a general process that drives assembly of many membrane-less cellular compartments. And we summarise recent evidence that phase separation of junctional scaffold proteins is involved in the assembly of tight junctions and focal adhesions.     

Biogenesis of inner membrane proteins in Escherichia coli

For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon. However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane. Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E. coli.     

Sec-translocase mediated membrane protein biogenesis

Alpha-helical transmembrane proteins in bacteria are localized within the plasma membrane. The membrane assembly of these proteins requires protein devices for insertion into the lipid bilayer. In E. coli, membrane proteins require the SRP pathway components Ffh, 4.5S RNA and FtsY for membrane targeting and the SecYEGDF translocase and, in some cases, SecA, for translocation of hydrophilic domains. In addition, YidC, a recently discovered membrane protein, mediates the membrane integration and folding of hydrophobic domains of membrane proteins. In this review, we will describe the current status of the protein targeting and membrane integration pathways.     

Heavy metal transport by the CusCFBA efflux system

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi‐protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu⁺ and Ag⁺ ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function.     

The role of lipids in membrane insertion and translocation of bacterial proteins

Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process.     

Distal regulatory regions restrict the expression of cis-linked genes to the tapetal cells

Rapid light-dependent turnover of the chloroplast-encoded D1 protein maintains photosystem II (PS II) functional over a wide range of light intensities. Following initiation of psbA mRNA translation, the elongating D1 is targeted, possibly by chloroplast signal recognition particle 54 (cpSRP54), to the thylakoid cpSecY translocation channel. Transmembrane domains of nascent D1 start interacting with other PS II core proteins already during the translocation process to ensure an efficient assembly of the multiprotein membrane complex. Here we review the progress recently made concerning the synthesis, targeting, membrane insertion and assembly to PS II of the chloroplast-encoded D1 protein and discuss the possible convergence of targeting and translocation of chloroplast- and nuclear-encoded thylakoid proteins.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

Protein trafficking and maturation regulate intramembrane proteolysis

Intramembrane-cleaving proteases (I-CLiPs) are membrane embedded proteolytic enzymes. All substrates identified so far are also membrane proteins, involving a number of critical cellular signaling as well as human diseases. After synthesis and assembly at the endoplasmic reticulum, membrane proteins are exported to the Golgi apparatus and transported to their sites of action. A number of studies have revealed the importance of the intracellular membrane trafficking in i-CLiP-mediated intramembrane proteolysis, not only for limiting the unnecessary encounter between i-CLiPs and their substrate but also for their cleavage site preference. In this review, we will discuss recent advances in our understanding of how each i-CLiP proteolysis is regulated by intracellular vesicle trafficking. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Mechanisms for quality control of misfolded transmembrane proteins

To prevent the accumulation of misfolded and aggregated proteins, the cell has developed a complex network of cellular quality control (QC) systems to recognize misfolded proteins and facilitate their refolding or degradation. The cell faces numerous obstacles when performing quality control on transmembrane proteins. Transmembrane proteins have domains on both sides of a membrane and QC systems in distinct compartments must coordinate to monitor the folding status of the protein. Additionally, transmembrane domains can have very complex organization and QC systems must be able to monitor the assembly of transmembrane domains in the membrane. In this review, we will discuss the QC systems involved in repair and degradation of misfolded transmembrane proteins. Also, we will elaborate on the factors that recognize folding defects of transmembrane domains and what happens when misfolded transmembrane proteins escape QC and aggregate. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Multiple pathways for the targeting of thylakoid proteins in chloroplasts

The assembly of the photosynthetic apparatus requires the import of numerous cytosolically synthesised proteins and their correct targeting into or across the thylakoid membrane. Biochemical and genetic studies have revealed the operation of several targeting pathways for these proteins, some of which are used for thylakoid lumen proteins whereas others are utilised by membrane proteins. Some pathways can be traced back to the prokarytoic ancestors of chloroplasts but at least one pathway appears to have arisen in response to the transfer of genes from the organelle to the nucleus. In this article we review recent findings in this field that point to the operation of a mechanistically unique protein translocase in both plastids and bacteria, and we discuss emerging data that reconcile the remarkable variety of targeting pathways with the natures of the substrate precursor proteins.     

In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force.

To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB). MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease. Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+. Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly. In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation. Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins. These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.     

Caveolae: Formation,dynamics, and function

Caveolae are abundant surface pits formed by the assembly of cytoplasmic proteins on a platform generated by caveolin integral membrane proteins and membrane lipids. This membranous assembly can bud off into the cell or can be disassembled releasing the cavin proteins into the cytosol. Disassembly can be triggered by increased membrane tension, or by stress stimuli, such as UV. Here, we discuss recent mechanistic studies showing how caveolae are formed and how their unique properties allow them to function as multifunctional protective and signaling structures.     

Review: nuclear lamins--structural proteins with fundamental functions

The nuclear lamina is located between the inner nuclear membrane and the peripheral chromatin. It is composed of both peripheral and integral membrane proteins, including lamins and lamina-associated proteins. Lamins can interact with one another, with lamina-associated proteins, with nuclear scaffold proteins, and with chromatin. Likewise, most of the lamina-associated proteins are likely to interact directly with chromatin. The nuclear lamina is required for proper cell cycle regulation, chromatin organization, DNA replication, cell differentiation, and apoptosis. Mutations in proteins of the nuclear lamina can disrupt these activities and cause genetic diseases. The structure and assembly of the nuclear lamina proteins and their roles in chromatin organization and cell cycle regulation were recently reviewed. In this review, we discuss the roles of the nuclear lamina in DNA replication and apoptosis and analyze how mutations in nuclear lamina proteins might cause genetic diseases.     

Membrane protein misassembly in disease

Helix-helix interactions play a central role in the folding and assembly of integral α-helical membrane proteins and are fundamentally dictated by the amino acid sequence of the TM domain. It is not surprising then that missense mutations that target these residues are often linked to disease. In this review, we focus on the molecular mechanisms through which missense mutations lead to aberrant folding and/or assembly of these proteins, and then discuss pharmacological approaches that may potentially mitigate or reverse the negative effects of these mutations. Improving our understanding of how missense mutations affect the interactions between TM α-helices will increase our capability to develop effective therapeutic approaches to counter the misassembly of these proteins and, ultimately, disease. This article is part of a Special Issue entitled: Protein Folding in Membranes.