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PubMed2005

Role of a conserved glutamate residue in the Escherichia coli SecA ATPase mechanism.

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the "DEVD" Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s(-1) versus 27 s(-1), respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439-17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis.

Adenosine Triphosphatases↗

PubMed2005

Subunits of the translocon interact with components of the oligosaccharyl transferase complex.

Following initiation of translocation across the membrane of the endoplasmic reticulum via the translocon, polypeptide chains are N-glycosylated by the oligosaccharyl transferase (OT) enzyme complex. Translocation and N-glycosylation are concurrent events and would be expected to require juxtaposition of the translocon and the OT complex. To determine whether any of the subunits of the OT complex and translocon mediate interactions between the two complexes, we initiated a systematic study in budding yeast using the split-ubiquitin approach. Interestingly, the OT subunit Stt3p was found to interact only with Sec61p, whereas another OT subunit, Ost4p, was found to interact with all three components of the translocon, Sec61p, Sbh1p, and Sss1p. The OT subunit Wbp1p was found to interact very strongly with Sec61p and Sbh1p and weakly with Sss1p. Other OT subunits, Ost1p, Ost2p, and Swp1p were found to interact with Sec61p and either Sbh1p or Sss1p. Ost3p exhibited a weak interaction with Sec61p and Sbh1p, whereas Ost5p and Ost6p interacted very weakly with Sec61p and failed to interact with Sbh1p or Sss1p. We were able to confirm these split-ubiquitin findings by a chemical cross-linking technique. Based on our findings using these two techniques, we conclude that the association of these two complexes is stabilized via multiple protein-protein contacts. Based on extrapolation of the structural parameters of the crystal structure of the prokaryotic Sec complex to the eukaryotic complex, we propose a working model to understand the organization of the translocon-OT supercomplex.

Cross-Linking Reagents↗

PubMed2005

Covalently dimerized SecA is functional in protein translocation.

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.

Adenosine Triphosphatases↗

PubMed2005

Proteolytic activity of HtpX, a membrane-bound and stress-controlled protease from Escherichia coli.

Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.

Adenosine Triphosphate↗

PubMed2005

The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA.

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (Kd approximately 10(-5) m). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an alpha-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.

Adenosine Triphosphatases↗

PubMed2005

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane.

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.

Adenosine Triphosphatases↗

PubMed2005

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli.

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.

Adenosine Triphosphatases↗

PubMed2006

Alternate recruitment of signal recognition particle and trigger factor to the signal sequence of a growing nascent polypeptide.

Different from cytoplasmic membrane proteins, presecretory proteins of bacteria usually do not require the signal recognition particle for targeting to the Sec translocon. Nevertheless signal sequences of presecretory proteins have been found in close proximity to signal recognition particle immediately after they have emerged from the ribosome. We show here that at the ribosome, the molecular environment of a signal sequence depends on the nature of downstream sequence elements that can cause an alternate recruitment of signal recognition particle and the ribosome-associated chaperone Trigger factor to a growing nascent chain. While signal recognition particle and Trigger factor might remain bound to the same ribosome, both ligands are clearly able to displace each other from a nascent chain. The data also imply that a signal sequence owes its molecular environment to the fact that it remains closely apposed to the ribosomal exit site during growth of a nascent secretory protein.

Adenosine Triphosphatases↗

PubMed2006

Subunit a of cytochrome o oxidase requires both YidC and SecYEG for membrane insertion.

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that facilitate the insertion of membrane proteins. Depletion of YidC in E. coli leads to a specific defect in the functional assembly of major energy transducing complexes such as the F1F0 ATPase and cytochrome bo3 oxidase. Here we report on the in vitro reconstitution of the membrane insertion of the CyoA subunit of cytochrome bo3 oxidase. Efficient insertion of in vitro synthesized pre-CyoA into proteoliposomes requires YidC, SecYEG, and SecA and occurs independently of the proton motive force. These data demonstrate that pre-CyoA is a substrate of a novel pathway that involves both SecYEG and YidC.

Cell Membrane↗

PubMed2006

The long alpha-helix of SecA is important for the ATPase coupling of translocation.

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

Adenosine Triphosphatases↗

PubMed2006

A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations.

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Bacterial Outer Membrane Proteins↗

PubMedDate not supplied

Bacterial secretome: the assembly manual and operating instructions (Review).

Bacterial protein secretion is a complex multi-stage reaction that is central to membrane and cell wall biosynthesis and essential for cell viability. An impressive array of experimental tools have been used to dissect this reaction into discreet sub-reactions. Synthesis of these data reveals a fascinating cascade of inter- and intra-molecular interactions that select, sort and target secretory polypeptides to the membrane and then spend metabolic energy to bias their vectorial movement across the membrane plane through a lipid-inaccessible proteinaceous environment. Transmembrane crossing is catalyzed by protein translocase, an astonishingly dynamic molecular machine. The unusual molecular features of the Sec pathway components allows a handful of proteins to catalyze the export of hundreds of secretory polypeptide substrates with astonishing fidelity. Knowledge of the molecular details of the secretion pathway allows us to rationally exploit these features in heterologous protein production biotechnologies and in the development of novel antibiotics.

Adenosine Triphosphatases↗

PubMed2004

The phylogeny and signature sequences characteristics of Fibrobacteres, Chlorobi, and Bacteroidetes.

Fibrobacteres, Chlorobi, and Bacteroidetes (FCB group) comprise three main bacterial phyla recognized on the basis of 16S rRNA trees. Presently, there are no distinctive biochemical or molecular characteristics known that can distinguish these bacteria from other bacterial phyla. The relationship of these bacteria to other phyla is also not known. This review describes many signatures, consisting of defined and conserved inserts in widely distributed proteins, that provide distinctive molecular markers for these groups of bacteria. These signatures serve to clarify the evolutionary relationship between members of the FCB group, and to other bacterial phyla. A 4 aa insert in DNA Gyrase B (GyrB) and a 45 aa insert in the SecA proteins are uniquely shared by various Bacteroidetes species. The insert in GyrB is present in all Bacteroidetes species (>100) covering different orders and families, indicating that it is a distinctive characteristic of the group. Three signatures consisting of an 18 aa insert in ATPase alpha-subunit, an 8-9 aa insert in the FtsK protein and a 1 aa insert in the UvrB protein are commonly shared only by the Bacteroidetes and Chlorobi homologs providing evidence that these two groups are specifically related to each other. Two additional inserts in the RNA polymerase beta'-subunit (5-7 aa) and Serine hydroxymethyl-transferase (14-16 aa), which are commonly present in various Bacteroidetes, Chlorobi, and Fibrobacteres homologs, but not any other bacteria, provide evidence that these groups shared a common ancestor exclusive of all other bacteria. The FCB groups of bacteria are indicated to have diverged from this common ancestor in the following order: Fibrobacteres --> Chlorobi --> Bacteriodetes. The inferences from signature sequences are strongly supported by phylogenetic analyses. These observations suggest that the FCB groups of bacteria should be placed in a single phylum rather than three distinct phyla. Signature sequences in a number of other proteins provide evidence that the FCB group of bacteria diverged at a similar time as the Chlamydiae group, and that the Spirochetes and Aquificales groups are its closest relatives.

Adenosine Triphosphatases↗

PubMed1997

Cloning and sequencing of the secY gene homolog from Mycobacterium bovis BCG.

The complete nucleotide sequence of a 1,513 bp fragment of Mycobacterium bovis BCG containing the secY gene homolog and partial adk gene that encodes an adenylate kinase has been determined. The secY gene of BCG has an open reading frame of 441 amino acids with homology to the SecY protein family. Comparative analyses of the deduced amino acid sequence of additional partial ORF revealed strong similarity to the known adenylate kinases.

Amino Acid Sequence↗

PubMed2002

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins.

Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner.

Adrenal Cortex↗

PubMed1993

Sec61p is adjacent to nascent type I and type II signal-anchor proteins during their membrane insertion.

We have identified membrane components which are adjacent to type I and type II signal-anchor proteins during their insertion into the membrane of the ER. Using two different cross-linking approaches a 37-38-kD nonglycosylated protein, previously identified as P37 (High, S., D. Görlich, M. Wiedmann, T. A. Rapoport, and B. Dobberstein. 1991. J. Cell Biol. 113:35-44), was found adjacent to all the membrane inserted nascent chains used in this study. On the basis of immunoprecipitation, this ER protein was shown to be identical to the recently identified mammalian Sec61 protein. Thus, Sec61p is the principal cross-linking partner of both type I and type II signal-anchor proteins during their membrane insertion (this work), and of secretory proteins during their translocation (Görlich, D., S. Prehn, E. Hartmann, K.-U. Kalies, and T. A. Rapoport. 1992. Cell. 71:489-503). We propose that membrane proteins of both orientations, and secretory proteins employ the same ER translocation sites, and that Sec61p is a core component of these sites.

Base Sequence↗

PubMed1994

Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex.

The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.

Animals↗

PubMed1995

Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.

Base Sequence↗