Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.     

Getting on target: the archaeal signal recognition particle

Protein translocation begins with the efficient targeting of secreted and membrane proteins to complexes embedded within the membrane. In Eukarya and Bacteria, this is achieved through the interaction of the signal recognition particle (SRP) with the nascent polypeptide chain. In Archaea, homologs of eukaryal and bacterial SRP-mediated translocation pathway components have been identified. Biochemical analysis has revealed that although the archaeal system incorporates various facets of the eukaryal and bacterial targeting systems, numerous aspects of the archaeal system are unique to this domain of life. Moreover, it is becoming increasingly clear that elucidation of the archaeal SRP pathway will provide answers to basic questions about protein targeting that cannot be obtained from examination of eukaryal or bacterial models. In this review, recent data regarding the molecular composition, functional behavior and evolutionary significance of the archaeal signal recognition particle pathway are discussed.     

Translocation of long chain fatty acids across the plasma membrane – lipid rafts and fatty acid transport proteins

Translocation of long chain fatty acids across the plasma membrane is achieved by a concert of co-existing mechanisms. These lipids can passively diffuse, but transport can also be accelerated by certain membrane proteins as well as lipid rafts. Lipid rafts are dynamic assemblies of proteins and lipids, that float freely within the two dimensional matrix of the membrane bilayer. They are receiving increasing attention as devices that regulate membrane function in vivo and play an important role in membrane trafficking and signal transduction. In this review we will discuss how lipid rafts might be involved in the uptake process and how the candidate proteins for fatty acid uptake FAT/CD36 and the FATP proteins interact with these domains. We will also discuss the functional role of FATPs in general. To our understanding FATPs are indirectly involved in the translocation process across the plasma membrane by providing long chain fatty acid synthetase activity.     

Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells

The vast majority of extracellular proteins are exported from mammalian cells by the endoplasmic reticulum/Golgi-dependent secretory pathway. For poorly understood reasons, however, a heterogenous group of extracellular proteins has been discovered that does not make use of signal peptide-dependent secretory transport. Both the release mechanisms and the molecular identity of the secretory machines involved have remained elusive. Recent studies now have established a subgroup of unconventional secretory proteins capable of translocating from the cytoplasm directly across the plasma membrane to get access to the exterior of eukaryotic cells. This review aims to focus on a detailed comparison of the subcellular site of membrane translocation of various unconventional secretory proteins such as the proangiogenic molecule fibroblast growth factor-2 (FGF-2) and Leishmania hydrophilic acylated surface protein B (HASP B). A potential link between membrane translocation and quality control as an integral part of unconventional secretory processes is discussed.     

Recognition of signal sequences

The hypothesis assumes that every continuous, entirely hydrophobic sequence of sufficient length, which is not involved in strong intramolecular contacts with other parts of the nascent protein chain, will function as a signal for translocation across the endoplasmic reticulum membrane or across the inner bacterial membrane. The signal peptide is proposed to be deeply immersed into a hydrophobic cleft of the receptor. Accordingly, only the entirely nonpolar peptides can be absorbed and, despite different primary structures, all of them would assume the same conformation dictated by the structure of the receptor pocket.     

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.     

Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane

Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.     

Polypeptide translocation across the endoplasmic reticulum membrane.

Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition. Much of the excitement in the next few years will come from the resolution of current controversies. What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction. Another approach is to identify homologues of these molecules in S. cerevisiae and to assess their importance in in vivo translocation. Several mechanistic questions remain to be addressed as well. Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years.     

Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli

Abstract: Fimbriae are long filamentous polymeric protein structures located at the surface of bacterial cells. They enable the bacteria to bind to specific receptor structures and thereby to colonise specific surfaces. Fimbriae consist of so-called major and minor subunits, which form, in a specific order, the fimbrial structure. In this review emphasis is put on the genetic organisation, regulation and especially on the biosynthesis of fimbriae of enterotoxigenic Escherichia coli strains, and more in particular on K88 and related fimbriae, with ample reference to the well-studied P and type 1 fimbriae. The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher ('doorkeeper'). Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with the periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the (ordered) translocation of the subunits across the outer membrane and their assembly into a grwoing fimbrial structure will be described. A model for K88 fimbriae is presented.     

Lipid involvement in protein translocation in Escherichia coli

Signal peptides play an essential role in protein translocation. This review summarizes the current knowledge of the structure of signal peptides and signal peptide-lipid interactions and addresses the possibility that signal peptide-lipid interactions initiate membrane translocation of precursor proteins. A new model for protein translocation in Escherichia coli is proposed, which includes as central features conformational changes of the signal peptide and signal-peptide-induced local changes in membrane organization (non-bilayer lipids).     

Shuttles and cycles: transport of proteins into the peroxisome matrix (review)

Peroxisomes are organelles that carry out diverse biochemical processes in eukaryotic cells, including the core pathways of beta-oxidation of lipid molecules and detoxification of reactive oxygen species. In multicellular organisms defects in peroxisome assembly result in multiple biochemical and developmental abnormalities. As peroxisomes do not contain genetic material, their protein content, and therefore function, is determined by the import of nuclearly encoded proteins from the cytosol and, presumably, removal of damaged or obsolete proteins. Import of matrix proteins can be broken down into four steps: targeting signal recognition by the cycling import receptors; receptor-cargo docking at the peroxisome membrane; translocation and cargo unloading; and receptor recycling. Import is mediated by a set of evolutionarily conserved proteins called peroxins that have been identified primarily via genetic screens, but knowledge of their biochemical activities remains largely unresolved. Recent studies have filled in some of the blanks regarding receptor recycling and the role of ubiquitination but outstanding questions remain concerning the nature of the translocon and its ability to accommodate folded, even oligomeric proteins, and the mechanism of cargo unloading and turnover of peroxisomal proteins. This review seeks to integrate recent findings from yeast, mammalian and plant systems to present an up to date account of how proteins enter the peroxisome matrix.     

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Membrane lipid rafts: new targets for immunoregulation

Engagement of immune receptors by antigen may lead to activation, cell proliferation, differentiation and effector functions. It has recently been proposed that the initiation and propagation of the signaling events taking place in immune cells occur in specialized membrane regions called lipid rafts. These detergent-insoluble glycolipid domains are specialized membrane compartments enriched in cholesterol and glycolipids. They also contain many lipid-modified signaling proteins such as tyrosine kinases of the Src family, GPI (glycosylphosphatidylinositol)-linked proteins as well as adaptor proteins. The confinement of signaling molecules in membrane subdomains suggests that lipid rafts function as platforms for the formation of multicomponent transduction complexes. Indeed, upon receptor binding, immune receptors become raft-associated and additional components of the signaling pathways are recruited to rafts in order to form signaling complexes. It has been speculated that the entry of immune receptors into rafts can regulate cell activation. Accordingly, numerous experiments have provided substantial evidence that raft integrity is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, Th1 versus Th2 lymphocytes) and sensitive to pharmacological agents. The aim of the present review is to summarize the current knowledge of immune receptor signal transduction with particular emphasis on the role of membrane compartments in immune activation. Finally, experimental evidences indicating that these membrane structures may represent clinically relevant potential targets for immune regulation, will be discussed.     

Molecular sorting of proteins into the cisternal secretory pathway

Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes. Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins. Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization. A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs. Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed.     

The unconventional secretory machinery of fibroblast growth factor 2

Unconventional secretory proteins represent a subpopulation of extracellular factors that are exported from eukaryotic cells by mechanisms that do not depend on the endoplasmic reticulum and the Golgi complex. Various pathways have been implicated in unconventional secretion including those involving intracellular membrane-bound intermediates and others that are based on direct protein translocation across plasma membranes. Interleukin 1β (IL1β) and fibroblast growth factor 2 (FGF2) are classical examples of unconventional secretory proteins with IL1β believed to be present in intracellular vesicles prior to secretion. By contrast, FGF2 represents an example of a non-vesicular mechanism of unconventional secretion. Here, the author discusses the current knowledge about the molecular machinery being involved in FGF2 secretion. To reveal both differential and common requirements, this review further aims at a comprehensive comparison of this mechanism with other unconventional secretory processes. In particular, a potentially general role of tyrosine phosphorylation as a regulatory signal in unconventional protein secretion will be discussed.     

Phosphorylation regulates the assembly of chloroplast import machinery

Chloroplast function depends on the translocation of cytosolically synthesized precursor proteins into the organelle. The recognition and transfer of most precursor proteins across the outer membrane depend on a membrane inserted complex. Two receptor components of this complex, Toc34 and Toc159, are GTPases, which can be phosphorylated by kinases present in the hosting membrane. However, the physiological function of phosphorylation is not yet understood in detail. It is demonstrated that both receptors are phosphorylated within their G-domains. In vitro, the phosphorylation of Toc34 disrupts both homo- and heterodimerization of the G-domains as determined using a phospho-mimicking mutant. In endogenous membranes this mutation or phosphorylation of the wild-type receptor disturbs the association of Toc34, but not of Toc159 with the translocation pore. Therefore, phosphorylation serves as an inhibitor for the association of Toc34 with other components of the complex and phosphorylation can now be discussed as a mechanism to exchange different isoforms of Toc34 within this ensemble.     

Targeting of proteins to the twin-arginine translocation pathway

The twin-arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N-terminal signal sequence containing a highly conserved twin-arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation.     

Shuttles and cycles: transport of proteins into the peroxisome matrix (Review)

Peroxisomes are organelles that carry out diverse biochemical processes in eukaryotic cells, including the core pathways of β-oxidation of lipid molecules and detoxification of reactive oxygen species. In multicellular organisms defects in peroxisome assembly result in multiple biochemical and developmental abnormalities. As peroxisomes do not contain genetic material, their protein content, and therefore function, is determined by the import of nuclearly encoded proteins from the cytosol and, presumably, removal of damaged or obsolete proteins. Import of matrix proteins can be broken down into four steps: targeting signal recognition by the cycling import receptors; receptor-cargo docking at the peroxisome membrane; translocation and cargo unloading; and receptor recycling. Import is mediated by a set of evolutionarily conserved proteins called peroxins that have been identified primarily via genetic screens, but knowledge of their biochemical activities remains largely unresolved. Recent studies have filled in some of the blanks regarding receptor recycling and the role of ubiquitination but outstanding questions remain concerning the nature of the translocon and its ability to accommodate folded, even oligomeric proteins, and the mechanism of cargo unloading and turnover of peroxisomal proteins. This review seeks to integrate recent findings from yeast, mammalian and plant systems to present an up to date account of how proteins enter the peroxisome matrix.     

Toc64--a preprotein-receptor at the outer membrane with bipartide function

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.