Protein translocation across the peroxisomal membrane

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism, of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs, through a large membrane protein assembly.     

Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.     

From membranes to organelles: Emerging roles for dynamin-like proteins in diverse cellular processes

Dynamin is a GTPase mechanoenzyme most noted for its role in vesicle scission during endocytosis, and belongs to the dynamin family proteins. The dynamin family consists of classical dynamins and dynamin-like proteins (DLPs). Due to structural and functional similarities DLPs are thought to carry out membrane tubulation and scission in a similar manner to dynamin. Here, we discuss the newly emerging roles for DLPs, which include vacuole fission and fusion, peroxisome maintenance, endocytosis and intracellular trafficking. Specific focus is given to the role of DLPs in the budding yeast Saccharomyces cerevisiae because the diverse function of DLPs has been well characterized in this organism. Recent insights into DLPs may provide a better understanding of mammalian dynamin and its associated diseases.     

Membrane translocation of folded proteins

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.     

Proteomics in Trypanosoma cruzi--localization of novel proteins to various organelles

The completion of the genome sequence of Trypanosoma cruzi has been followed by several studies of protein expression, with the long-term aim to obtain a complete picture of the parasite proteome. We report a proteomic analysis of an organellar cell fraction from T. cruzi CL Brener epimastigotes. A total of 396 proteins were identified by LC-MS/MS. Of these, 138 were annotated as hypothetical in the genome databases and the rest could be assigned to several metabolic and biosynthetic pathways, transport, and structural functions. Comparative analysis with a whole cell proteome study resulted in the validation of the expression of 173 additional proteins. Of these, 38 proteins previously reported in other stages were not found in the only large-scale study of the total epimastigote stage proteome. A selected set of identified proteins was analyzed further to investigate gene copy number, sequence variation, transmembrane domains, and targeting signals. The genes were cloned and the proteins expressed with a c-myc epitope tag in T. cruzi epimastigotes. Immunofluorescence microscopy revealed the localization of these proteins in different cellular compartments such as ER, acidocalcisome, mitochondrion, and putative cytoplasmic transport or delivery vesicles. The results demonstrate that the use of enriched subcellular fractions allows the detection of T. cruzi proteins that are undetected by whole cell proteomic methods.     

Sorting and function of peroxisomal membrane proteins

Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.     

The isoelectric point of proteins influences their translocation to the extrahaustorial matrix of the barley powdery mildew fungus

Many biotrophic fungal plant pathogens develop feeding structures, haustoria, inside living plant cells, which are essential for their success. Extrahaustorial membranes (EHMs) surround haustoria and delimit the extrahaustorial matrices (EHMxs). Little is known about transport mechanisms across EHMs and what properties proteins and nutrients need in order to cross these membranes. To investigate this further, we expressed fluorescent proteins in the cytosol of infected barley leaf epidermal cells after particle bombardment and investigated properties that influenced their localisation in the powdery mildew EHMx. We showed that this translocation is favoured by a neutral isoelectric point (pI) between 6.0 and 8.4. However, for proteins larger than 50 kDa, pI alone does not explain their localisation, hinting towards a more complex interplay between pI, size, and sequence properties. We discuss the possibility that an EHM translocon is involved in protein uptake into the EHMx.     

Self-organization versus watchmaker: molecular motors and protein translocation

Generation of directional movement at the molecular scale is a phenomenon crucial for biological organization and dynamics. It is traditionally described in mechanistic terms, in consistency with the conventional machine-like image of the cell. The designated and highly specialized protein machines and molecular motors are presumed to bring about most of cellular motion. A review of experimental data suggests, however, that uncritical adherence to mechanistic interpretations may limit the ability of researchers to comprehend and model biology. Specifically, this article illustrates that the interpretation of molecular motors and protein translocation in terms of stochasticity and self-organization appears to provide a more adequate and fruitful conceptual framework for understanding of biological organization at the molecular scale.     

Characterization of the supporting role of SecE in protein translocation

SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic α-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open.     

以SecA蛋白为“动力泵”的细菌Sec蛋白转运途径

细菌细胞中,三分之一的蛋白质是在合成后被转运到细胞质外才发挥功能的.其中大多数蛋白是通过Sec途径(即分泌途径secretion pathway)进行跨膜运动的.Sec转运酶是一个多组分的蛋白质复合体,膜蛋白三聚体SecYEG及水解ATP的动力蛋白SecA构成了Sec转运酶的核心.整合膜蛋白SecD,SecF和vajC形成了一个复合体亚单位,可与SecYEG相连并稳定SecA蛋白的膜结合形式.SecB是蛋白质转运中的伴侣分子,可以和很多蛋白质前体结合.SecM是由位于secA基因上游的secM基因编码的,可调节SecA蛋白的合成量,维持细胞在不同环境条件下的正常生长.新生肽链的信号肽被高度保守的SRP特异性识别.伴侣分子SecB通过与细胞膜上的SecA二聚体特异性结合将蛋白质前体引导至Sec转运途径,起始转运过程.结合蛋白质前体的SecA与组成转运通道的SecYEG复合体具有较高的亲和性.SecA经历插入和脱离细胞内膜SecYEG通道的循环,为转运提供所需的能量,每一次循环可推动20多个氨基酸的连续跨膜运动.     

Evolution of the prokaryotic protein translocation complex: a comparison of archaeal and bacterial versions of SecDF

Protein translocation across the prokaryotic plasma membrane occurs at the translocon, an evolutionarily conserved membrane-embedded proteinaceous complex. Together with the core components SecYE, prokaryotic translocons also contain auxilliary proteins, such as SecDF. Alignment of bacterial and archaeal SecDF protein sequences reveals the presence of a similar number of homologous regions within each protein. Moreover, the conserved sequence domains in the archaeal proteins are located in similar positions as their bacterial counterparts. When these domains are, however, compared along Bacteria-Archaea lines, a much lower degree of similarity is detected. In Bacteria, SecDF are thought to modulate the membrane association of SecA, the ATPase that provides the driving force for bacterial protein secretion. As no archaeal version of SecA has been detected, the sequence differences reported here may reflect functional differences between bacterial and archaeal SecDF proteins, and by extension, between the bacterial and archaeal protein translocation processes. Moreover, the apparent absence of SecDF in several completed archaeal genomes suggests that differences may exist in the process of protein translocation within the archaeal domain itself.     

The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production

The biotechnological production of recombinant proteins is challenged by processes that decrease the yield, such as protease action, aggregation, or misfolding. Today, the variation of strains and vector systems or the modulation of inducible promoter activities is commonly used to optimize expression systems. Alternatively, aggregation to inclusion bodies may be a desired starting point for protein isolation and refolding. The discovery of the twin-arginine translocation (Tat) system for folded proteins now opens new perspectives because in most cases, the Tat machinery does not allow the passage of unfolded proteins. This feature of the Tat system can be exploited for biotechnological purposes, as expression systems may be developed that ensure a virtually complete folding of a recombinant protein before purification. This review focuses on the characteristics that make recombinant Tat systems attractive for biotechnology and discusses problems and possible solutions for an efficient translocation of folded proteins.     

Protein translocationin vitro: Biochemical characterization of genetically defined translocation components

Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation inE. coli. The powerful combination of these approaches is exemplified in the use of anin vitro protein synthesis-protein translocaltion system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of thesecA, secB, andsecY gene products and the demonstration of their requirement forin vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or protein-ATPase.     

Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology

Peroxisomes are cell organelles bounded by a single membrane with a basically oxidative metabolism. Peroxisomes house catalase and H₂O₂‐producing flavin‐oxidases as the main protein constituents. However, since their discovery in early fifties, a number of new enzymes and metabolic pathways have been reported to be also confined to these organelles. Thus, the presence of exo‐ and endo‐peptidases, superoxide dismutases, the enzymes of the plant ascorbate‐glutathione cycle plus ascorbate and glutathione, several NADP‐dehydrogenases, and also L‐arginine‐dependent nitric oxide synthase activity has evidenced the relevant role of these organelles in cell physiology. In recent years, the study of new functions of peroxisomes has become a field of intensive research in cell biology, and these organelles have been proposed to be a source of important signal molecules for different transduction pathways. In plants, peroxisomes participate in seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species (ROS and RNS, respectively). In order to decipher the nature and specific role of the peroxisomal proteins in these processes, several approaches including in vivo and in vitro import assays and generation of mutants have been used. In the last decade, the development of genomics and the report of the first plant genomes provided plant biologists a powerful tool to assign to peroxisomes those proteins which harbored any of the two peroxisomal targeting signals (PTS, either PTS1 or PTS2) described so far. Unfortunately, those molecular approaches could not give any response to those proteins previously localized in plant peroxisomes by classical biochemical and cell biology methods that did not contain any PTS. However, more recently, proteomic studies of highly purified organelles have provided evidence of the presence in peroxisomes of new proteins not previously reported. Thus, the contribution of proteomic approaches to the biology of peroxisomes is essential, not only for elucidation of the mechanisms involved in the import of the PTS1‐ and PTS2‐independent proteins, but also to the understanding of the role of these organelles in the cell physiology of plant growth and development.     

Channel crossing: how are proteins shipped across the bacterial plasma membrane?

The structure of the first protein-conducting channel was determined more than a decade ago. Today, we are still puzzled by the outstanding problem of protein translocation—the dynamic mechanism underlying the consignment of proteins across and into membranes. This review is an attempt to summarize and understand the energy transducing capabilities of protein-translocating machines, with emphasis on bacterial systems: how polypeptides make headway against the lipid bilayer and how the process is coupled to the free energy associated with ATP hydrolysis and the transmembrane protein motive force. In order to explore how cargo is driven across the membrane, the known structures of the protein-translocation machines are set out against the background of the historic literature, and in the light of experiments conducted in their wake. The paper will focus on the bacterial general secretory (Sec) pathway (SecY-complex), and its eukaryotic counterpart (Sec61-complex), which ferry proteins across the membrane in an unfolded state, as well as the unrelated Tat system that assembles bespoke channels for the export of folded proteins.     

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins

Subcellular organelles in eukaryotes are surrounded by lipid membranes.In an endomembrane system,vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles.However,organellar proteins for chloroplasts,mitochondria,the nucleus,and peroxisomes that are translated in the cytosol are directly imported into their target organelles.Chloroplasts are a plant-specific organelle with outer and inner envelope membranes,a dual-membrane structure that is simil...     

Changes in levels of pancreatic endoplasmic reticulum proteins that function in translocation and maturation of secretory proteins in response to cholecystokinin

Two pathways operate to target newly-synthesised proteins to the endoplasmic reticulum. In one, the signal recognition particle attaches to the signal sequences of nascent chains on ribosomes and slows or stops translation until contact is made with the docking protein at the membrane. The second operates via molecular chaperons. The pathways converge at the level of a 43 kDa signal binding protein integrated into the membrane, where translocation through a proteinaceous pore is initiated. In the lumen, proteins fold and disulphide formation is catalysed by the enzyme protein disulphide isomerase. The heavy chain binding protein may attach to unassembled or unfolded proteins and prevent their exit from the ER to the Golgi. Cholecystokinin (CCK) treatment increases the biosynthesis and secretion of pancreatic proteins, increases the levels of PDI and the 43 kDa binding protein, and reduces levels of BiP. These proteins may be possible targets for genetic manipulation to improve processing of heterologous proteins from cultured mammalian cells.     

Surface-associated proteins of Staphylococcus aureus: Their possible roles in virulence

Abstract A class of proteins that are associated with the cell surface of Gram-positive bacteria has been recognised. Common structural features which are implicated in the proper secretion and attachment of these proteins to the cell surface occur in the C-termini. N-terminal domains interact with the host by binding to soluble host proteins, to matrix proteins or to host cells. They probably have important roles in pathogenicity by allowing bacteria to avoid host defences and by acting as adhesins. Four such proteins of Staphylococcus aureus have been characterised: protein A (immunoglobulin binding protein), fibronectin binding proteins, collagen binding protein and the fibrinogen binding protein (clumping factor). Site-specific mutants are being used to define their roles in pathogenesis in in vitro and in vivo models of adherence and infection.