Awaking TIM22, a dynamic ligand-gated channel for protein insertion in the mitochondrial inner membrane

Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.     

An artificial transmembrane segment directs SecA, SecB, and electrochemical potential-dependent translocation of a long amino-terminal tail

Many integral membrane proteins contain an amino-terminal segment, often referred to as an N-tail, that is translocated across a membrane. In many cases, translocation of the N-tail is initiated by a cleavable, amino-terminal signal peptide. For N-tail proteins lacking a signal peptide, translocation is initiated by a transmembrane segment that is carboxyl to the translocated segment. The mechanism of membrane translocation of these segments, although poorly understood, has been reported to be independent of the protein secretion machinery. In contrast, here we describe alkaline phosphatase mutants containing artificial transmembrane segments that demonstrate that translocation of a long N-tail across the membrane is dependent upon SecA, SecB, and the electrochemical potential in the absence of a signal peptide. The corresponding mutants containing signal peptides also use the secretion machinery but are less sensitive to inhibition of its components. We present evidence that inhibition of SecA by sodium azide is incomplete even at high concentrations of inhibitor, which suggests why SecA-dependent translocation may not have been detected in other systems. Furthermore, by varying the charge around the transmembrane segment, we find that in the absence of a signal peptide, the orientation of the membrane-bound alkaline phosphatase is dictated by the positive inside rule. However, the presence of a signal peptide is an overriding factor in membrane orientation and renders all mutants in an Nout-Cin orientation.     

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.     

Signal recognition particle (SRP)-mediated targeting and Sec-dependent translocation of an extracellular Escherichia coli protein

Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion, i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.     

Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae

Chimeras created by fusing the monomeric red fluorescent protein (RFP) to a bacterial lipoprotein signal peptide (lipoRFPs) were visualized in the cell envelope by epifluorescence microscopy. Plasmolysis of the bacteria separated the inner and outer membranes, allowing the specific subcellular localization of lipoRFPs to be determined in situ. When equipped with the canonical inner membrane lipoprotein retention signal CDSR, lipoRFP was located in the inner membrane in Escherichia coli, whereas the outer membrane sorting signal CSSR caused lipoRFP to localize to the outer membrane. CFSR-RFP was also routed to the outer membrane, but CFNSR-RFP was located in the inner membrane, consistent with previous data showing that this sequence functions as an inner membrane retention signal. These four lipoproteins exhibited identical localization patterns in a panel of members of the family Enterobacteriaceae, showing that the lipoprotein sorting rules are conserved in these bacteria and validating the use of E. coli as a model system. Although most predicted inner membrane lipoproteins in these bacteria have an aspartate residue after the fatty acylated N-terminal cysteine residue, alternative signals such as CFN can and probably do function in parallel, as indicated by the existence of putative inner membrane lipoproteins with this sequence at their N termini.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

YidC-dependent translocation of green fluorescence protein fused to the FliP cleavable signal peptide

Escherichia coli FliP is a rare bacterial polytopic membrane protein synthesized with a cleavable, highly hydrophobic signal peptide. More hydrophilic Tat-dependent or Sec-dependent signal peptide is functionally capable of substituting for the FliP signal peptide, but a signal anchor of inner membrane protein fails to do so. To assess the intrinsic characteristics of the FliP signal peptide in mediating protein translocation, we fused it to green fluorescence protein and observed that the translocation of the chimera (FliPss-GFP) was dependent of Ffh, SecA, SecY and SecD. In addition, we showed for the first time the involvement of YidC in protein translocation across the inner membrane.     

Membrane targeting of a bacterial virulence factor harbouring an extended signal peptide

Filamentous haemagglutinin (FHA) is the major adhesin of Bordetella pertussis, the whooping cough agent. FHA is synthesized as a 367-kDa precursor harbouring a remarkably long signal peptide with an N-terminal extension that is conserved among related virulence proteins. FHA is secreted via the two-partner secretion pathway that involves transport across the outer membrane by a cognate transporter protein. Here we have analyzed the mechanism by which FHA is targeted to, and translocated across, the inner membrane. Studies were performed both in vitro using Escherichia coli inside-out inner membrane vesicles and in vivo by pulse-chase labelling of Bordetella pertussis cells. The data collectively indicate that like classical periplasmic and outer membrane proteins, FHA requires SecA and SecB for its export through the SecYEG translocon in the inner membrane. Although short nascent chains of FHA were found to cross-link to signal recognition particle (SRP), we did not obtain indication for an SRP-dependent, co-translational membrane targeting provoked by the FHA signal sequence. Our results rule out that the extended signal peptide of FHA determines a specific mode of membrane targeting but rather suggest that it might influence the export rate at the inner membrane.     

Import of chemically synthesized signal peptides into rat liver mitochondria

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.     

大肠杆菌的蛋白分泌机制

大肠杆菌的分泌蛋白定位于内膜、外膜、周质空间和胞外环境,它们在N端或C端带有一定的结构包含着分泌信号,这两类分泌蛋白在各自特定的一组蛋白因子的协助下跨越内膜,再通过目前尚不清楚的方式实现其最终定位.N端带有信号肽的分子在跨越内膜时得到Sec家族蛋白因子协助,信号肽在跨膜过程中可能被切除,该过程由ATP和电化学势提供能量.C端带分泌信号的分子主要受到Hly家族分子协助,一次穿过内膜和外膜而不经过周质空间.     

Conservative sorting in the muroplasts of Cyanophora paradoxa: a reevaluation based on the completed genome sequence

After primary endosymbiosis, massive gene transfer occurred from the genome of the cyanobacterial endosymbiont to the nucleus of the protist host cell. In parallel, a specific protein import apparatus arose for reimport of many, but not all products of the genes moved to the nuclear genome. Presequences evolved to allow recognition of plastid proteins at the envelope and their translocation to the stroma. However, plastids (and cyanobacteria) also comprise five other subcompartments. Protein sorting to the cyanobacterial thylakoid membrane, the thylakoid lumen, the inner envelope membrane, the periplasmic space, and the outer envelope membrane is achieved by prokaryotic protein translocases recognizing, e.g., signal sequences. The “conservative sorting” hypothesis postulates that these translocases remained functional in endosymbiotic organelles and obtained their passengers not only from imported proteins but also from proteins synthesized in organello. For proteins synthesized in the cytosol, a collaboration of the general import apparatus and the former prokaryotic translocase is necessary which is often reflected by the use of bipartite presequences, e.g., stroma targeting peptide and signal peptide. For plants, this concept has been experimentally proven and verified. The muroplasts from Cyanophora paradoxa, that have several features more in common with cyanobacteria than with plastids, were analyzed with the availability of the recently completed nuclear genome sequence. Interesting findings include the absence of the post-translational signal recognition particle pathway, dual Sec translocases in thylakoid and inner envelope membranes that are produced from a single set of genes, and a co-translational signal recognition pathway operating without a 4.5S RNA component.     

An amino-terminal signal sequence abrogates the intrinsic membrane-targeting information of mitochondrial uncoupling protein

Mitochondrial uncoupling protein, a polytopic integral protein of the inner membrane, is initially made in the cytoplasm as a soluble polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Earlier studies (Liu, X., Bell, A. W., Freeman, K. B., and Shore, G. C. (1988) J. Cell Biol. 107, 503-509) identified internal regions of the molecule that are critical for targeting and membrane insertion. Here, we demonstrate that the ability of uncoupling protein to insert into the inner membrane is abrogated when the molecule is fused behind the matrix-targeting signal of preornithine carbamyltransferase; the hybrid protein was imported across the inner membrane and deposited in the matrix where it was processed. In this context, however, the processed product remained in the matrix and was incapable of inserting into the inner membrane.     

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence,gliding motility and the degradation of complex biopolymers

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres‐Chlorobi‐Bacteroidetes superphylum. Proteins secreted by the T9SS have an N‐terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C‐terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C‐terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components.     

A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase

A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins.     

The phase property of membrane phospholipids is affected by the functionality of signal peptides from the Escherichia coli ribose-binding protein

We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers. The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition. The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species. The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid. The wild-type and revertant signal peptides predominantly formed alpha-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy. In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer. However, the alpha-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer. An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not. The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid. We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.     

Identification of cold-inducible inner membrane proteins of the psychrotrophic bacterium, Shewanella livingstonensis Ac10, by proteomic analysis

Shewanella livingstonensis Ac10 is a psychrotrophic Gram-negative bacterium that grows at temperatures close to 0°C. Previous proteomic studies of this bacterium identified cold-inducible soluble proteins and outer membrane proteins that could possibly be involved in its cold adaptation (Kawamoto et al. in Extremophiles 11:819–826, 2007). In this study, we established a method for separating the inner and outer membranes by sucrose density gradient ultracentrifugation and performed proteomic studies of the inner membrane fraction. The cells were grown at temperatures of 4 and 18°C, and phospholipid-enriched inner membrane fractions were obtained. Two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting analysis of the proteins identified 14 cold-inducible proteins (more than a 2-fold increase at 4°C). Six of these proteins were predicted to be inner membrane proteins. Two predicted periplasmic proteins, 5 predicted cytoplasmic proteins, and 1 predicted outer membrane protein were also found in the inner membrane fraction, suggesting their association with the inner membrane proteins and/or lipids. These cold-inducible proteins included proteins that are presumed to be involved in chemotaxis (AtoS and PspA), membrane protein biogenesis (DegP, SurA, and FtsY), and morphogenesis (MreB). These findings provide a basis for further studies on the cold-adaptation mechanism of this bacterium.     

Protease IV, a cytoplasmic membrane protein of Escherichia coli, has signal peptide peptidase activity

During export of the outer membrane lipoprotein across the cytoplasmic membrane, the signal peptide of the lipoprotein undergoes two successive proteolytic attacks, cleavage of the signal peptide by signal peptidase and digestion of the cleaved signal peptide by an enzyme called signal peptide peptidase(s) (Hussain, M., Ichihara, S., and Mizushima, S. (1982) J. Biol. Chem. 257, 5177-5182; Hussain, M., Ozawa, Y., Ichihara, S., and Mizushima, S. (1982) Eur. J. Biochem. 129, 233-239). Here we report that protease IV, a cytoplasmic membrane protease, exhibits the signal peptide peptidase activity. The signal peptide peptidase activity was cofractionated with protease IV throughout the entire process of purification of the latter enzyme. Only the signal peptide was digested by the peptidase among membrane proteins. Both the signal peptide peptidase activity and the protease IV activity were inhibited to similar degrees by antipain, leupeptin, chymostatin, and elastatinal that are known to inhibit the signal peptide peptidase activity in the cell envelope. From these results we conclude that protease IV is the signal peptide peptidase that is responsible for signal peptide digestion in the cytoplasmic membrane. The peptidase attacked the signal peptide only after its release from the precursor protein.     

Role of the signal sequence in proteorhodopsin biogenesis in E. coli

Blue-absorbing proteorhodopsin (BPR) from marine bacteria is a retinal-bound, light-activated, outwards proton transporter containing seven α-helical transmembrane segments (TMS). It is synthesized as a precursor species (pre-BPR) with a predicted N-terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre-BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre-BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild-type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial “pro” domain rather than a traditional secretory peptide.     

Investigating lipoprotein biogenesis and function in the model Gram‐positive bacterium Streptomyces coelicolor

Lipoproteins are a distinct class of bacterial membrane proteins that are translocated across the cytoplasmic membrane primarily by the Sec general secretory pathway and then lipidated on a conserved cysteine by the enzyme lipoprotein diacylglycerol transferase (Lgt). The signal peptide is cleaved by lipoprotein signal peptidase (Lsp) to leave the lipid‐modified cysteine at the N‐terminus of the mature lipoprotein. In all Gram‐positive bacteria tested to date this pathway is non‐essential and the lipid attaches the protein to the outer leaflet of the cytoplasmic membrane. Here we identify lipoproteins in the model Gram‐positive bacterium Streptomyces coelicolor using bioinformatics coupled with proteomic and downstream analysis. We report that Streptomyces species translocate large numbers of lipoproteins out via the Tat (twin arginine translocase) pathway and we present evidence that lipoprotein biogenesis might be an essential pathway in S. coelicolor. This is the first analysis of lipoproteins and lipoprotein biogenesis in Streptomyces and provides the first evidence that lipoprotein biogenesis could be essential in a Gram‐positive bacterium. This report also provides the first experimental evidence that Tat plays a major role in the translocation of lipoproteins in a specific bacterium.