Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.     

Translocation of nascent non-signal sequence protein in heated Escherichia coli

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.     

线粒体蛋白质的转运

线粒体含有约1000种蛋白质,其中99%由细胞核DNA编码,在细胞质核糖体上合成后被分别转运至线粒体的内膜或外膜上、基质或膜间隙中。由众多分子机器组成的线粒体蛋白质转运系统参与了该生物学过程的执行。线粒体DNA编码的13种蛋白质也由该系统转运至线粒体内膜。本文就线粒体蛋白质转运系统中线粒体前体蛋白质的定位分选信号、转运复合物和转运途径作简要介绍。     

Influence of membrane-lipid composition on translocation of nascent proteins in heated Escherichia coli

In studies using Escherichia coli we have shown that new protein species appear in the outer membrane fraction with concomitant losses of nascent proteins from the soluble and inner membrane fractions following heat exposure. Of the various explanations for this phenomenon, temperature-induced membrane disorganization appeared the most likely. It is suggested that heat mimics the action of the signal sequence of a protein on the lipid bilayer allowing non-signal-sequence-containing proteins to be translocated. To test this hypothesis we grew E. coli K1060 cells, an unsaturated fatty acid requiring auxotroph, supplemented during growth with fatty acids of varying chain length in an attempt to determine whether biological membranes of varying ability to maintain their bilayer configuration could be constructed. The rationale being that such membranes would allow us to determine whether differences in translocation would occur in cells grown at the same temperature supplemented with either 16:1 or 20:1 unsaturated fatty acids when the cells were subjected to a series of thermal insults. Protein translocation occurred to a greater extent and at lower temperatures in cells supplemented with the longer chain fatty acid. Treatment of outer membranes with either 1 M salt, 6 M urea or high pH and studies determining fluorescent polarization values by scanning up and down through a series of temperatures ranging from 15 to 49 degrees C indicate that the proteins translocated by heat to the outer membrane are integral. Protein translocation may represent an adaptive response to an altered environment enabling the cell to respond to stress by stabilizing its outer membrane.     

Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane

Leader peptidase cleaves the amino-terminal leader sequences of many secreted and membrane proteins. We have examined the function of leader peptidase by constructing an Escherichia coli strain where its synthesis is controlled by the arabinose B promoter. This strain requires arabinose for growth. When the synthesis of leader peptidase is repressed, protein precursors accumulate, including the precursors of M13 coat protein (an inner membrane protein), maltose binding protein (a periplasmic protein), and OmpA protein (an outer membrane protein). These precursors are translocated across the plasma membrane, as judged by their sensitivity to added proteinase K. However, pro-OmpA and pre-maltose binding protein are retained at the outer surface of the inner membrane. Thus, leader peptides anchor translocated pre-proteins to the outer surface of the plasma membrane and must be removed to allow their subsequent release into the periplasm or transit to the outer membrane.     

Structural elements of the mitochondrial preprotein-conducting channel Tom40 dissolved by bioinformatics and mass spectrometry

Most mitochondrial proteins are imported into mitochondria from the cytosolic compartment. Proteins destined for the outer or inner membrane, the inter-membrane space, or the matrix are recognized and translocated by the TOM machinery containing the specialized protein import channel Tom40. The latter is a protein with β-barrel shape, which is suggested to have evolved from a porin-type protein. To obtain structural insights in the absence of a crystal structure the membrane topology of Tom40 from Neurospora crassa was determined by limited proteolysis combined with mass spectrometry. The results were interpreted on the basis of a structural model that has been generated for NcTom40 by using the structure of mouse VDAC-1 as a template and amino acid sequence information of approximately 270 different Tom40 and approximately 480 VDAC amino acid sequences for refinement. The model largely explains the observed accessible cleavage sites and serves as a structural basis for the investigation of physicochemical properties of the ensemble of our Tom40 sequence data set. By this means we discovered two conserved polar slides in the pore interior. One is possibly involved in the positioning of a pore-inserted helix; the other one might be important for mitochondrial pre-sequence peptide binding as it is only present in Tom40 but not in VDAC proteins. The outer surface of the Tom40 barrel reveals two conserved amino acid clusters. They may be involved in binding other components of the TOM complex or bridging components of the TIM machinery of the mitochondrial inner membrane.     

Cytosolic aggregates in presence of non‐translocated proteins perturb endoplasmic reticulum structure and dynamics

Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non‐translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non‐translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three‐way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins—reticulons, and altered distribution of those associated with the three‐way ER junctions—Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non‐translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven‐pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non‐translocated precursor proteins—their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.     

Early events in yeast mitochondrial protein targeting

Protein import into mitochondria involves a number of complex steps occurring in the cytosol, on the mitochondrial surface, and inside the organelle. Once an initial interaction between mitochondrial proteins and their specific receptors occurs, the proteins are transported into the organelle in a series of reactions involving (in the case of a protein to be translocated into the mitochondrial matrix) the mitochondrial membrane potential, ATP hydrolysis and an undetermined number of membrane components. Inside the organelle, mitochondrial proteins are processed and sorted to their final intramitochondrial destinations. The earliest steps in the import process take place in the cytosol and include the synthesis of the mitochondrial proteins themselves, their interaction with cytosolic factors, and perhaps the establishment of cotranslational import complexes on the mitochondrial surface. These early events are important because it is during this phase that the system as a whole is most sensitive to cytosolic conditions that may exert control over the entire import process.     

Travelling of proteins through membranes: translocation into chloroplasts

 Most proteins involved in plastid biogenesis are encoded by the nuclear genome. They are synthesised in the cytosol and have to be transported toward and subsequently translocated into the organelle. This targeting and import process is initiated by a specific chloroplast-targeting signal. The targeting signal of the preprotein is recognised and modified by cytosolic proteins which function in transport toward the chloroplast and in maintaining the import-competent state of the preprotein. The precursor is transferred onto a multi-component complex in the outer envelope of the chloroplasts, which is formed by receptor proteins and the translocation channel. Some proteins, not containing transit sequences, are directly sorted into the outer membrane whereas the majority, containing transit sequences, will be translocated into the stroma. This involves the joint action of a protein complex in the outer envelope, one complex in the inner envelope, and soluble proteins in the intermembrane space and the stroma. The origin of this translocation complex following the endosymbiotic events is an unsolved question. Recent identification of homologous proteins to some members of this machinery in the cyanobacterium Synechocystis PCC6803 gives an initial insight into the origin of the translocation complex. Received: 27 December 1999 / Accepted: 29 March 2000     

Direct membrane insertion of voltage-dependent anion-selective channel protein catalyzed by mitochondrial Tom20.

Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent beta-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.     

Dietary lipids and aging compromise chaperone-mediated autophagy by similar mechanisms

Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.     

Dietary lipids and aging compromise chaperone-mediated autophagy by similar mechanisms

Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Mitochondrial protein import

Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.     

Quantitative and spatio‐temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re‐localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re‐targeted to alternative sites such as the inner membrane in the presence of site‐specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage‐free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.     

Trehalose is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum but not for maintenance of membrane traffic functions after severe heat stress

Saccharomyces cerevisiae cells grown at physiological temperature 24 degrees C require preconditioning at 37 degrees C to acquire tolerance towards brief exposure to 48-50 degrees C. During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance. It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat-damaged proteins in the yeast cytosol. We have demonstrated that heat-denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature. Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen. Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress. In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat-damaged glycoproteins to bioactive and secretion-competent forms failed or was retarded. In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic. However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.     

Cytosolic events involved in chloroplast protein targeting

Chloroplasts are unique organelles that are responsible for photosynthesis. Although chloroplasts contain their own genome, the majority of chloroplast proteins are encoded by the nuclear genome. These proteins are transported to the chloroplasts after translation in the cytosol. Chloroplasts contain three membrane systems (outer/inner envelope and thylakoid membranes) that subdivide the interior into three soluble compartments known as the intermembrane space, stroma, and thylakoid lumen. Several targeting mechanisms are required to deliver proteins to the correct chloroplast membrane or soluble compartment. These mechanisms have been extensively studied using purified chloroplasts in vitro. Prior to targeting these proteins to the various compartments of the chloroplast, they must be correctly sorted in the cytosol. To date, it is not clear how these proteins are sorted in the cytosol and then targeted to the chloroplasts. Recently, the cytosolic carrier protein AKR2 and its associated cofactor Hsp17.8 for outer envelope membrane proteins of chloroplasts were identified. Additionally, a mechanism for controlling unimported plastid precursors in the cytosol has been discovered. This review will mainly focus on recent findings concerning the possible cytosolic events that occur prior to protein targeting to the chloroplasts. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.     

Cytokinesis in bacteria.

Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.     

Role of DegP for two-partner secretion in Bordetella

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In Gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in β-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis , represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long β-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37°C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro , DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.