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PubMed2001

Complementation of bacterial SecE by a chloroplastic homologue.

The SecE protein is an essential component of the SecAYE-translocase, which mediates protein translocation across the cytoplasmic membrane in bacteria. In the thylakoid membranes of chloroplasts, a protein homologous to SecE, chloroplastic (cp) SecE, has been identified. However, the functional role of cpSecE has not been established experimentally. In this report we show that cpSecE in cells depleted for bacterial SecE (i) supports growth, (ii) stabilizes, just like bacterial SecE, the Sec-translocase core component SecY, and (iii) supports Sec-dependent protein translocation. This indicates that cpSecE can functionally replace bacterial SecE in vivo, and strongly suggests that the thylakoid membrane contains a SecAYE-like translocase with functional and structural similarities to the bacterial complex. This study further underscores the evolutionary link between chloroplasts and bacteria.

Adenosine Triphosphatases↗
PubMed2001

YidC/Oxa1p/Alb3: evolutionarily conserved mediators of membrane protein assembly.

This review focuses on a novel, evolutionarily conserved mediator of membrane protein assembly in bacteria, mitochondria and chloroplasts. This factor is designated YidC in Escherichia coli, and is localized in the inner membrane. YidC is homologous to Oxa1p in the mitochondrial inner membrane and Alb3 in the chloroplast thylakoid membrane, but does not seem to have a homologue in the endoplasmic reticulum membrane. It has been suggested that YidC operates both as a separate unit and in connection with the SecYEG-translocon depending on the substrate membrane protein that is integrated into the membrane. Mitochondria do not possess a SecYEG-like complex and Oxa1p is thought to form, or to contribute to the formation of, a novel translocon in the mitochondrial inner membrane. Alb3 in the chloroplast thylakoid membrane is, just like YidC and Oxa1p, involved in membrane protein assembly, but only few details are known.

Arabidopsis Proteins↗
PubMed2002

SecY-SecY and SecY-SecG contacts revealed by site-specific crosslinking.

Protein translocation across the cytoplasmic membrane of Escherichia coli is mediated by the integral membrane complex SecYEG and the peripherally bound ATPase SecA. To probe the environment of the cytoplasmic domains of SecY within the SecYEG complex, we introduced single cysteine residues in each of the six cytoplasmic domains. Neighbouring SecY molecules with a single cysteine residue in cytoplasmic domains C1, C2 or C6 formed a disulfide bond upon oxidation. The presence of the disulfide bond between two C2 domains reversibly inhibited protein translocation. Chemical crosslinking showed that the C2 and C3 domains are in close proximity of SecG and chemical modification of the cysteine residue in the C5 domain with N-ethyl-maleimide or fluorescein-5-maleimide inactivates the SecYEG complex. Taken together, our data give novel insights in the interactions between subunits of the SecYEG complex and emphasise the importance of cytoplasmic domain C5 for SecY functioning.

Amino Acid Sequence↗
PubMed2003

Depletion of SecDF-YajC causes a decrease in the level of SecG: implication for their functional interaction.

SecA and an apparatus comprising SecYEG and SecDF-YajC complexes catalyze protein translocation across the Escherichia coli membrane. SecDF-YajC and SecG facilitate membrane insertion of SecA, which is the driving force for protein translocation. Here we report that SecDF-YajC depletion together with SecG depletion nearly completely inhibits protein translocation both in vivo and in vitro, although SecDF-YajC had been thought to be unnecessary for in vitro translocation. The level of SecG in membranes decreased to about half upon SecDF-YajC depletion and recovered to a normal level when SecDF-YajC was expressed. SecDF-YajC inhibited disulfide bond formation between two SecG molecules possessing a single cysteine residue. These results suggest functional interaction between SecDF-YajC and SecG.

Antigens, Bacterial↗
PubMed1997

Subunit interactions in the Escherichia coli protein translocase: SecE and SecG associate independently with SecY.

We used hexahistidine-tagged SecE and SecY to study how the core subunits (SecY, SecE and SecG) of Escherichia coli protein translocase interact with each other. Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+-NTA agarose affinity binding. Although His6-SecE, expressed in wild-type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells. His6-SecY brought down both SecE and SecG, as expected. Interestingly, His6-SecY24 was able to bring down SecG but not SecE. These results confirm our previous conclusion that the secY24 alteration impairs the SecY-SecE interaction, and demonstrate that SecY and SecG can form a complex that does not contain SecE. Likewise, SecY-SecE complex could be isolated from the secG-deleted strain. The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26 degrees C, whereas the SecY-SecE complex without SecG dissociated at a slightly lower temperature (20-23 degrees C). We conclude that each of SecE and SecG independently binds to SecY, the central subunit of protein translocase, although the trimeric complex is more stable than the binary complexes.

Bacterial Proteins↗
PubMed1997

In vitro membrane integration of leader peptidase depends on the Sec machinery and anionic phospholipids and can occur post-translationally.

A cell-free system based on a lysate and membrane vesicles from Escherichia coli is used to study characteristics of the membrane integration reaction of the polytopic membrane protein leader peptidase (Lep). Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease. Integration is most efficient when coupled to translation but can also occur post-translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids. Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro.

Adenosine Triphosphatases↗
PubMed1997

Interaction of SecB with soluble SecA.

The preprotein binding molecular chaperone SecB functions by preventing the premature folding of the preprotein in the cytosol, and targeting it to the peripheral subunit SecA of the translocase at the cytoplasmic membrane. The nature of the interaction of SecB with soluble SecA was studied by fluorescence anisotropy spectroscopy of Ru(bpy)2(dcbpy)-labeled SecA in the presence of increasing concentrations of SecB. A more than 50-fold difference in affinity for the cytosolic SecA compared to translocase associated SecA seems to prevent unproductive binding of SecB to the cytosolic SecA and stresses its targeting function.

Adenosine Triphosphatases↗
PubMed1998

Escherichia coli SecA shape and dimensions.

SecA shape and conformational flexibility in solution were studied by small angle X-ray scattering. Dimeric SecA is a very elongated molecule, 15 nm long and 8 nm wide. SecA is therefore four times as long as the membrane is wide. The two globular protomers are distinctly separated and share limited surface of intermolecular contacts. ATP, ADP or adenylyl-imidodiphosphate (AMP-PNP) binding does not alter the SecA radius of gyration. A SecA mutant that catalyzes multiple rounds of ATP hydrolysis does not undergo conformational changes detectable by small angle X-ray scattering (SAXS). We conclude that SecA conformational alterations observed biochemically during nucleotide interaction are only small-scale and localized. The ramifications of these findings on SecA/SecYEG interaction are discussed.

Adenosine Diphosphate↗
PubMed1998

Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptides to the Sec61 complex.

Nascent polypeptide associated complex (NAC) interacts with nascent polypeptides emerging from ribosomes. Both signal recognition particle (SRP) and NAC work together to ensure specificity in co-translational targeting by competing for binding to the ribosomal membrane attachment site. While SRP selects signal-containing ribosomes for targeting, NAC prevents targeting of signal peptide-less nascent chains to the endoplasmic reticulum membrane. Here we show that the ribosome binding that occurs in NAC's absence delivers signalless nascent chains to the Sec61 complex, underscoring the danger of unregulated exposure of the ribosomal M-site. Recently, the idea that NAC prevents ribosome binding has been challenged. By carefully examining the physiologic NAC concentration in a variety of tissues from different species we here demonstrate that the discrepancy resulted from subphysiologic NAC concentrations.

Animals↗
PubMed1999

Oligomeric complexes involved in translocation of proteins across the membrane of the endoplasmic reticulum.

Proteins involved in protein translocation across the membrane of the endoplasmic reticulum assemble into different oligomeric complexes depending on their state of function. To analyse such membrane protein complexes we fractionated proteins of mammalian rough microsomes and analysed them using blue native PAGE and immunoblotting. Among the proteins characterised are the Sec61p complex, the oligosaccharyl transferase (OST) complex, the translocon-associated protein (TRAP) complex, the TRAM and RAMP4 proteins, the signal recognition particle (SRP) and the SRP receptor (SR). Interestingly, the RAMP4 protein, SR and OST complex display more than one oligomeric form.

Animals↗
PubMed1999

Thermodynamics of nucleotide binding to NBS-I of the Bacillus subtilis preprotein translocase subunit SecA.

SecA is the dissociatable nucleotide and preprotein binding subunit of the bacterial translocase. The thermodynamics of nucleotide binding to soluble SecA at nucleotide binding site I were determined by isothermal titration calorimetry. Binding of ADP and non-hydrolyzable ATPgammaS is enthalpy-driven (DeltaH(0) of -14.44 and -5.56 kcal/mol, respectively), but is accompanied by opposite entropic contributions (DeltaS(0) of -18.25 and 9.55 cal/mol/K, respectively). ADP binding results in a large change in the heat capacity of SecA (DeltaC(p)=-780 cal/mol/K). It is suggested that ADP binding promotes the interaction between the two thermodynamically discernible domains of SecA which is accompanied by a shielding of hydrophobic surface from solvent.

Adenosine Diphosphate↗
PubMed1999

Ricin A chain utilises the endoplasmic reticulum-associated protein degradation pathway to enter the cytosol of yeast.

Cytotoxic proteins such as ricin A chain (RTA) have target substrates in the cytosol and therefore have to reach this cellular compartment in order to act. RTA is thought to translocate into the cytosol from the lumen of the endoplasmic reticulum (ER), although how it traverses the ER membrane has not been established. Using yeast mutants defective in various aspects of the ER-associated protein degradation (ERAD) pathway, we show that RTA introduced into the yeast ER subverts this pathway to enter the cytosol via the Sec61p translocon. A significant proportion of the exported RTA avoided proteasomal degradation. These data are consistent with the contention that the RTA component from ricin endocytosed by mammalian cells may likewise exploit ERAD to translocate into the cytosol.

Biological Transport↗
PubMed1999

The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E. coli-based protein production.

OmpT, an outer membrane-localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification. SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro. Here we report that SecY cleavage occurs even in cell extracts from ompT-disrupted cells, if they carry an F plasmid derivative. A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis. These results indicate that the absence of an F-like plasmid should be checked when choosing a host strain for E. coli-based protein production.

Bacterial Outer Membrane Proteins↗
PubMed2002

Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH.

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.

Adenosine Triphosphatases↗
PubMed2003

Nucleotide binding induces changes in the oligomeric state and conformation of Sec A in a lipid environment: a small-angle neutron-scattering study.

In Escherichia coli, SecA is a large, multifunctional protein that is a vital component of the general protein secretion pathway. In its membrane-bound form it functions as the motor component of the protein translocase, perhaps through successive rounds of membrane insertion and ATP hydrolysis. To understand both the energy conversion process and translocase assembly, we have used contrast-matched, small-angle neutron-scattering (SANS) experiments to examine SecA in small unilamellar vesicles of E.coli phospholipids. In the absence of nucleotide, we observe a dimeric form of SecA with a radius of gyration comparable to that previously observed for SecA in solution. In contrast, the presence of either ADP or a non-hydrolyzable ATP analog induces conversion to a monomeric form. The larger radius of gyration for the ATP-bound relative to the ADP-bound form suggests the former has a more expanded global conformation. This is the first direct structural determination of SecA in a lipid bilayer. The SANS data indicate that nucleotide turnover can function as a switch of conformation of SecA in the membrane in a manner consistent with its proposed role in successive cycles of deep membrane penetration and release with concommitant preprotein insertion.

Adenosine Triphosphatases↗
PubMed2001

Protein traffic in bacteria: multiple routes from the ribosome to and across the membrane.

Bacteria use several routes to target their exported proteins to the plasma membrane. The majority are exported through pores formed by SecY and SecE. Two different molecular machineries are used to target proteins to the SecYE translocon. Translocated proteins, synthesized as precursors with cleavable signal sequences, require cytoplasmic chaperones, such as SecB, to remain competent for posttranslational transport. In concert with SecB, SecA targets the precursors to SecY and energizes their translocation by its ATPase activity. The latter function involves a partial insertion of SecA itself into the SecYE translocon, a process that is strongly assisted by a couple of membrane proteins, SecG, SecD, SecF, YajC, and the proton gradient across the membrane. Integral membrane proteins, however, are specifically recognized by a direct interaction between their noncleaved signal anchor sequences and the bacterial signal recognition particle (SRP) consisting of Ffh and 4.5S RNA. Recognition occurs during synthesis at the ribosome and leads to a cotranslational targeting to SecYE that is mediated by FtsY and the hydrolysis of GTP. No other Sec protein is required for integration unless the membrane protein also contains long translocated domains that engage the SecA machinery. Discrimination between SecA/SecB- and SRP-dependent targeting involves the specificity of SRP for hydrophobic signal anchor sequences and the exclusion of SRP from nascent chains of translocated proteins by trigger factor, a ribosome-associated chaperone. The SecYE pore accepts only unfolded proteins. In contrast, a class of redox factor-containing proteins leaves the cell only as completely folded proteins. They are distinguished by a twin arginine motif of their signal sequences that by an unknown mechanism targets them to specific pores. A few membrane proteins insert spontaneously into the bacterial plasma membrane without the need for targeting factors and SecYE. Insertion depends only on hydrophobic interactions between their transmembrane segments and the lipid bilayer and on the transmembrane potential. Finally, outer membrane proteins of Gram-negative bacteria after having crossed the plasma membrane are released into the periplasm, where they undergo distinct folding events until they insert as trimers into the outer membrane. These folding processes require distinct molecular chaperones of the periplasm, such as Skp, SurA, and PpiD.

Adenosine Triphosphatases↗