Translocon Complex Research Articles

Sec61α is required for dorsal closure duringDrosophilaembryogenesis through its regulation of Dpp signaling

During dorsal closure in Drosophila, signaling events in the dorsalmost row of epidermal cells (DME cells) direct the migration of lateral epidermal sheets towards the dorsal midline where they fuse to enclose the embryo. A Jun amino-terminal kinase (JNK) cascade in the DME cells induces the expression of Decapentaplegic (Dpp). Dpp signaling then regulates the cytoskeleton in the DME cells and amnioserosa to affect the cell shape changes necessary to complete dorsal closure. We identified a mutation in Sec61alpha that specifically perturbs dorsal closure. Sec61alpha encodes the main subunit of the translocon complex for co-translational import of proteins into the ER. JNK signaling is normal in Sec61alpha mutant embryos, but Dpp signaling is attenuated and the DME cells fail to maintain an actinomyosin cable as epithelial migration fails. Consistent with this model, dorsal closure is rescued in Sec61alpha mutant embryos by an activated form of the Dpp receptor Thick veins.

EseD, a Putative T3SS Translocon Component of Edwardsiella tarda, Contributes to Virulence in Fish and is a Candidate for Vaccine Development

Edwardsiella tarda has a type III secretion system (T3SS) essential for pathogenesis. EseD, together with EseB and EseC, has been suggested to form a putative T3SS translocon complex, although its further function is unclear. To investigate the physiological role of EseD, a mutant strain of E. tarda was constructed with an in-frame deletion of the entire eseD gene. One finding was that the ∆eseD mutant decreased the secretion levels of EseC and EseB proteins. Additionally, the ∆eseD mutant showed attenuated swarming and contact-hemolysis abilities. However, the ∆eseD mutant showed increased biofilm formation. Complementation of the mutant strain with eseD restored these phenotypes to those similar to the wild-type strain. Furthermore, infection experiments in fish showed that the ∆eseD mutant exhibited slower proliferation and a tenfold decrease in virulence in fish. These results indicate a specific role of EseD in the pathogenesis of E. tarda. Finally, recombinant EseD protein elicited high antibody titers in immunized fish and various levels of protection against lethal challenge with the wild-type strain. These results indicate that EseD protein may be a candidate antigen for development of a subunit vaccine against Edwardsiellosis.

Yersinia Translocon Complexes are Stabilized in Nanolipoprotein Particles (NLPs)

To enter host cells and evade host defenses, many gram-negative bacteria, including the plague pathogen, Yersinia pestis, utilize proteins that are able to interact with and enter host membranes. One particular mechanism for invasion is the type III secretion system, which is a highly ordered complex for injecting bacterial proteins into host cells, using a complex referred to as a translocon pore. Our results show cell-free expression of YopB and YopD was enhanced in the presence of liposomes or NLPs. However, liposomes containing YopB/D tended to aggregate and precipitate. In order to enable the study of the type III secretion proteins we have applied cell-free approaches for producing soluble Y. pestis membrane associated proteins YopB and YopD that are involved in the translocon pore as a complex supported by nanolipoprotein particles (NLPs). With addition of NLP, the YopB/D complex was rendered soluble. AFM showed that soluble YopB/D complex was associated with NLPs as measured by a height increase compared to NLPs not containing YopB/D. Preliminary AFM results also demonstrated binding between LcrV and YopB/D-NLPs which is indicative of proper folding in the NLP structure. Interaction studies of the YopB/D translocon embedded in a membrane with effectors such as LcrV may elucidate the poorly understood pore-forming event that helps this pathogen to evade the host defenses. Our method is applicable to other membrane proteins and represents a unique solution to solubility and purification problems.

Presenilin‐2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+ uptake

We have previously shown that familial Alzheimer’s disease mutants of presenilin-2 (PS2) and, to a lesser extent, of presenilin-1 (PS1) lower the Ca2+ concentration of intracellular stores. We here examined the mechanism by which wild-type and mutant PS2 affect store Ca2+ handling. By using HeLa, SH-SY5Y and MEFs as model cells, and recombinant aequorins as Ca2+ probes, we show evidence that transient expression of either wild-type or mutant PS2 increases the passive Ca2+ leakage: both ryanodine- and IP3-receptors contribute to Ca2+ exit out of the ER, whereas the ribosome translocon complex is not involved. In SH-SY5Y cells and MEFs, wild-type and mutant PS2 potently reduce the uptake of Ca2+ inside the stores, an effect that can be counteracted by over-expression of SERCA-2B. On this line, in wild-type MEFs, lowering the endogenous level of PS2 by RNA interference, increases the Ca2+-loading capability of intracellular stores. Furthermore, we show that in PS double knockout MEFs, reduction of Ca2+ stores is mimicked by the expression of PS2-D366A, a loss-of-function mutant, uncleaved because also devoid of presenilinase activity but not by co-expression of the two catalytic active fragments of PS2. In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca2+ uptake by intracellular stores. To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast ofPlasmodium falciparum

Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been "rewired" to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.

Cellular mechanisms of membrane protein folding.

The membrane protein-folding problem can be articulated by two central questions. How is protein topology established by selective peptide transport to opposite sides of the cellular membrane? And how are transmembrane segments inserted, integrated and folded within the lipid bilayer? In eukaryotes, this process usually takes place in the endoplasmic reticulum, coincident with protein synthesis, and is facilitated by the translating ribosome and the Sec61 translocon complex (RTC). At its core, the RTC forms a dynamic pathway through which the elongating nascent polypeptide moves as it is delivered into the cytosolic, lumenal and lipid compartments. This Perspective will focus on emerging evidence that the RTC functions as a protein-folding machine that restricts conformational space by establishing transmembrane topology and yet provides a permissive environment that enables nascent transmembrane domains to efficiently progress down their folding energy landscape.

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of approximately 1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction. Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD(50) by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

A Conserved Hydrophobic Tetrad near the C Terminus of the Secretory Na+-K+-2Cl- Cotransporter (NKCC1) Is Required for Its Correct Intracellular Processing

Little is known about the intracellular folding and trafficking of integral membrane proteins. Here we identify a hydrophobic amino acid tetrad (ILLV) close to the C terminus of the secretory Na+-K+-2Cl- cotransporter (NKCC1) that is important for the proper intracellular processing of this protein. This tetrad appears in a C-terminal sequence pattern that is conserved across species in a number of members of the NKCC1 gene family (slc12) of electroneutral salt transporters. We studied the effects of various mutations of these amino acids on NKCC1 transiently transfected into HEK-293 cells. Our results show that mutation of two of these residues to alanine leads to a >50% reduction in expression and complex glycosylation levels and that multiple mutations to alanine have cumulative effects. By contrast, scrambling of these amino acids, or mutation of other nearby conserved C-terminal residues, has little effect on these parameters. Mutation of ILLV to AAAA reduces complex glycosylation of NKCC1 by approximately 90% and results in a protein that does not form stable dimers and is retained in the endoplasmic reticulum in a highly aggregated state. Our results are consistent with the hypothesis that mutation of the hydrophobic tetrad ILLV to AAAA leads to the ab initio misfolding and concomitant aggregation of this NKCC1 mutant, resulting in its retention in the endoplasmic reticulum.

Protein transport in organelles: The Toc complex way of preprotein import

Most of the estimated 1000 or so chloroplast proteins are synthesized as cytosolic preproteins with N-terminal cleavable targeting sequences (transit peptide). Translocon complexes at the outer (Toc) and inner chloroplast envelope membrane (Tic) concertedly facilitate post-translational import of preproteins into the chloroplast. Three components, the Toc34 and Toc159 GTPases together with the Toc75 channel, form the core of the Toc complex. The two GTPases act as GTP-dependent receptors at the chloroplast surface and promote insertion of the preprotein across the Toc75 channel. Additional factors guide preproteins to the Toc complex or support their stable ATP-dependent binding to the chloroplast. This minireview describes the components of the Toc complex and their function during the initial steps of preprotein translocation across the chloroplast envelope.

Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins

The universally conserved SecYEG/Sec61 translocon constitutes the major protein-conducting channel in the cytoplasmic membrane of bacteria and the endoplasmic reticulum membrane of eukaryotes. It is engaged in both translocating secretory proteins across the membrane as well as in integrating membrane proteins into the lipid phase of the membrane. In the current study we have detected distinct SecYEG translocon complexes in native Escherichia coli membranes. Blue-Native-PAGE revealed the presence of a 200-kDa SecYEG complex in resting membranes. When the SecA-dependent secretory protein pOmpA was trapped inside the SecYEG channel, a smaller SecY-containing complex of approximately 140-kDa was observed, which probably corresponds to a monomeric SecYEG-substrate complex. Trapping the SRP-dependent polytopic membrane protein mannitol permease in the SecYEG translocon, resulted in two complexes of 250 and 600 kDa, each containing both SecY and the translocon-associated membrane protein YidC. The appearance of both complexes was correlated with the number of transmembrane domains that were exposed during targeting of mannitol permease to the membrane. These results suggest that the assembly or the stability of the bacterial SecYEG translocon is influenced by the substrate that needs to be transported.

In vivo interaction between atToc33 and atToc159 GTP-binding domains demonstrated in a plant split-ubiquitin system

The GTPases atToc33 and atToc159 are pre-protein receptor components of the translocon complex at the outer chloroplast membrane in Arabidopsis. Despite their participation in the same complex in vivo, evidence for their interaction is still lacking. Here, a split-ubiquitin system is engineered for use in plants, and the in vivo interaction of the Toc GTPases in Arabidopsis and tobacco protoplasts is shown. Using the same method, the self-interaction of the peroxisomal membrane protein atPex11e is demonstrated. The finding suggests a more general suitability of the split-ubiquitin system as a plant in vivo interaction assay.

Analysis of Glycosylation Site Occupancy Reveals a Role for Ost3p and Ost6p in Site-specific N-Glycosylation Efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.

P4-203: Full-length and not mature presenilin 2 increases the leakage of intracellular calcium stores: Specific mechanisms aand targets

Background: We have previously shown that fibroblasts from FAD patients carrying mutations in either PS1 (M146L, P117L) or PS2 (M239I, T122R) showed a reduced instead of an exaggerated calcium release from intracellular stores (1-3). These and other PS1/2 mutants (PS1-A246E, PS1-L286V, PS2-N141I), as well as the wild type (wt) forms of these proteins share the same feature when transiently over-expressed in different cell lines and rat primary neurons. Whereas PS1 mutants cause modest reductions under transient expression, PS2 mutants dramatically reduce the store calcium content. Accordingly, the latter also cause lower steady-state calcium concentrations at the level of the endoplasmic reticulum (ER) and Golgi apparatus (3). Methods: By employing both fura-2 and organelletargeted aequorins to monitor calcium concentrations in the cytosolic and the lumen, we here investigate in HeLa, SH-SY5Y and MEFseither wt or devoid of endogenous PSs (DKO MEFs) the mechanisms by which PS2 variants alter store calcium handling. Results: We provide evidence that: i) expression of wt or mutant PS2 reduces the store calcium content both by increasing the ER calcium leakage and by reducing the ER calcium uptake by SERCA pumps; ii) the full-length (FL) but not the mature form of the protein is directly involved in store calcium handling; iii) the effect is completely independent of g-secretase activity since it is not rescued by pre-incubation with enzyme inhibitors or by expression of a loss-of-function mutant (PS2-D366A); iv) the ryanodine receptor and the ribosome translocon complex are not directly involved. Whether FL PS2 variants represent by themselves a leakage pathway (4) or whether their effect is mediated by IP3 receptors is now under investigation. Conclusions: We suggest that, at variance with the majority of FL PS1 mutants that leave unchanged or even overload intracellular calcium stores, making the cells more susceptible to toxic stimuli, FL PS2 mutants, by lowering ER/Golgi calcium levels result in a less aggressive FAD phenotypes.

Identification of Two Translocon Proteins of Vibrio parahaemolyticus Type III Secretion System 2

The type III secretion system (T3SS) translocon complex is composed of several associated proteins, which form a translocation channel through the host cell plasma membrane. These proteins are key molecules that are involved in the pathogenicity of many T3SS-positive bacteria, because they are necessary to deliver effector proteins into host cells. A T3SS designated T3SS2 of Vibrio parahaemolyticus is thought to be related to the enterotoxicity of this bacterium in humans, but the effector translocation mechanism of T3SS2 is unclear because there is only one gene (the VPA1362 gene) in the T3SS2 region that is homologous to other translocon protein genes. It is also not known whether the VPA1362 protein is functional in the translocon of T3SS2 or whether it is sufficient to form the translocation channel of T3SS2. In this study, we identified both VPA1362 (designated VopB2) and VPA1361 (designated VopD2) as T3SS2-dependent secretion proteins. Functional analysis of these proteins showed that they are essential for T3SS2-dependent cytotoxicity, for the translocation of one of the T3SS2 effector proteins (VopT), and for the contact-dependent activity of pore formation in infected cells in vitro. Their targeting to the host cell membrane depends on T3SS2, and furthermore, they are necessary for T3SS2-dependent enterotoxicity in vivo. These results indicate that VopB2 and VopD2 act as translocon proteins of V. parahaemolyticus T3SS2 and hence have a critical role in the T3SS2-dependent enterotoxicity of this bacterium.

A Pentapeptide Motif Related to a Pigment Binding Site in the Major Light-Harvesting Protein of Photosystem II, LHCII, Governs Substrate-Dependent Plastid Import of NADPH:Protochlorophyllide Oxidoreductase A

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) A is the only known example thus far of a nucleus-encoded plastid protein that is imported to its final destination in a substrate-dependent, Pchlide-regulated manner. Previous work has shown that the cytosolic PORA precursor (pPORA) does not utilize the general import site but uses a distinct translocon designated the Pchlide-dependent translocon complex. Here we demonstrate that a pentapeptide motif, threonine-threonine-serine-proline-glycine (TTSPG) in pPORA's transit peptide (transA), is involved in Pchlide-dependent transport. Deletion of this motif from the COOH-terminal end of transA abolished both Pchlide binding and protein import. Incorporation of the TTSPG motif into normally non-Pchlide-responsive transit sequences conferred the pigment binding properties onto the engineered chimeric precursors but was insufficient to render protein import substrate dependent. An additional motif was identified in the NH(2)-terminal part of transA that was needed for binding of the precursor to the Pchlide-dependent translocon complex. Point mutations of the TTSPG motif identified glycine as the Pchlide binding site. By analogy to the major light-harvesting chlorophyll a/b binding protein of photosystem II, we propose that the peptidyl carbonyl oxygen of glycine may bind directly or via a water molecule to the central Mg atom of the pigment.

Prediction of the translocon-mediated membrane insertion free energies of protein sequences

Helical membrane proteins (HMPs) play crucial roles in a variety of cellular processes. Unlike water-soluble proteins, HMPs need not only to fold but also get inserted into the membrane to be fully functional. This process of membrane insertion is mediated by the translocon complex. Thus, it is of great interest to develop computational methods for predicting the translocon-mediated membrane insertion free energies of protein sequences. We have developed Membrane Insertion (MINS), a novel sequence-based computational method for predicting the membrane insertion free energies of protein sequences. A benchmark test gives a correlation coefficient of 0.74 between predicted and observed free energies for 357 known cases, which corresponds to a mean unsigned error of 0.41 kcal/mol. These results are significantly better than those obtained by traditional hydropathy analysis. Moreover, the ability of MINS to reasonably predict membrane insertion free energies of protein sequences allows for effective identification of transmembrane (TM) segments. Subsequently, MINS was applied to predict the membrane insertion free energies of 316 TM segments found in known structures. An in-depth analysis of the predicted free energies reveals a number of interesting findings about the biogenesis and structural stability of HMPs. A web server for MINS is available at http://service.bioinformatik.uni-saarland.de/mins

The translocon and the non-specific transport of small molecules in the endoplasmic reticulum (Review)

Transmembrane fluxes are major determinants of several enzyme activities localized in the luminal compartment of the endoplasmic reticulum (ER). Although a large number of metabolites were shown to be transported across the ER membrane, only a few transporters have been identified so far. It can be assumed that the basal permeability of ER membrane vesicles (microsomes) to a variety of small molecules is due to the presence of a low-selectivity channel or pore rather than many strictly specific transporters. The translocon complex is a possible candidate for this role because it transitionally forms an open channel in the ER membrane and an increasing amount of evidence shows the permeation of small compounds through this channel. It seems plausible that the translocon pore is not only responsible for inward and outward peptide translocation but also contributes to basal Ca(2+) leakage from the ER and ensures the substrate supply for certain luminal ER enzymes.

Mutations in the Sec61p Channel Affecting Signal Sequence Recognition and Membrane Protein Topology

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.

Climp-63-mediated binding of microtubules to the ER affects the lateral mobility of translocon complexes

Microtubules are frequently seen in close proximity to membranes of the endoplasmic reticulum (ER), and the membrane protein CLIMP-63 is thought to mediate specific interaction between these two structures. It was, therefore, of interest to investigate whether these microtubules are in fact responsible for the highly restricted lateral mobility of the translocon complexes in M3/18 cells as described before. As determined by fluorescence recovery after photobleaching, the breakdown of microtubules caused by drug treatment or by overexpression of the microtubule-severing protein spastin, resulted in an increased lateral mobility of the translocons that are assembled into polysomes. Also, the expression of a CLIMP-63 mutant lacking the microtubule-binding domain resulted in a significant increase of the lateral mobility of the translocon complexes. The most striking increase in the diffusion rate of the translocon complexes was observed in M3/18 cells transfected with a siRNA that effectively knocked down the expression of the endogenous CLIMP-63. It appears, therefore, that interaction of microtubules with the ER results in the immobilization of translocon complexes that are part of membrane-bound polysomes, and may play a role in the mechanism that segregates the rough and smooth domains of the ER.