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W Wickner

Publications and source records attributed to W Wickner.

At least 73 records · Page 4Linked to original sources
PubMed1991

Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase.

Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA.

Adenosine Triphosphatases↗
PubMed1990

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.

Bacterial Outer Membrane Proteins↗
PubMed1990

The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation.

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.

Adenosine Triphosphatases↗
PubMed1990

The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins.

The ATPase activity of SecA is stimulated by E. coli plasma membrane vesicles bearing SecY protein and a precursor protein such as proOmpA. This activity is termed "translocation ATPase". Liposomes alone can also stimulate SecA ATPase, but membrane proteins block this stimulation in native inner membranes. We define the stimulation of SecA ATPase by lipid as "SecA/lipid ATPase". SecA/lipid ATPase, translocation ATPase, and translocation into inner membrane vesicles require acidic phospholipids, suggesting an underlying unity of mechanism. ProOmpA and ATP stabilize liposome-bound SecA. Full SecA/lipid ATPase activity and stability are also seen when a mixture of a leader peptide and either OmpA or maltose binding protein (MBP) are added instead of proOmpA, while neither the leader peptide alone nor OmpA or MBP suffice. Cytosolic proteins in conjuction with a leader peptide are less active in this reaction, indicating that liposome-bound SecA protein recognizes both leader and mature domains.

Adenosine Triphosphatases↗
PubMed1990

ProOmpA contains secondary and tertiary structure prior to translocation and is shielded from aggregation by association with SecB protein.

Escherichia coli protein export involves cytosolic components termed molecular chaperones which function to stabilize precursors for membrane translocation. It has been suggested that chaperones maintain precursor proteins in a loosely folded state. We now demonstrate that purified proOmpA in its translocation component conformation contains both secondary and tertiary structure as analyzed by circular dichroism and intrinsic tryptophan fluorescence. Association with one molecular chaperone, SecB, subtly modulates the conformation of proOmpA and stabilizes it by inhibiting aggregation, permitting its translocation across inverted E.coli inner membrane vesicles. These results suggest that translocation competence does not simply result from the maintenance of an unfolded state and that molecular chaperones can stabilize precursor proteins by inhibiting their oligomerization.

Bacterial Outer Membrane Proteins↗
PubMed1990

Solubilization and functional reconstitution of the protein-translocation enzymes of Escherichia coli.

The SecY protein and other membrane proteins of Escherichia coli were solubilized by mixed micelles of n-octyl beta-D-glucopyranoside, phospholipids, and glycerol. Proteoliposomes formed from this extract by detergent dialysis supported energy-dependent translocation and processing of pro-OmpA. Translocation required ATP, SecY, and SecA and was stimulated by a proton-motive force. These results provide an important assay for the isolation and identification of membrane components involved in protein translocation.

Bacterial Outer Membrane Proteins↗
PubMed1990

Trigger factor depletion or overproduction causes defective cell division but does not block protein export.

Trigger factor is an abundant cytosolic protein of Escherichia coli which can stabilize proOmpA for in vitro translocation across inner membrane vesicles. The gene encoding E. coli trigger factor was isolated and sequenced, allowing construction of strains in which the expression of trigger factor is readily regulated. We found no defect in the in vivo rate of synthesis or secretion of proOmpA in trigger factor-depleted cells. The primary physiological defect in trigger factor-depleted or -overproducing cells is an enrichment of filamented cells. Filamentation of the trigger factor-overproducing strain is suppressed by a multicopy plasmid expressing the essential division gene ftsZ, suggesting that trigger factor has an important role in cell division.

Amino Acid Isomerases↗
PubMed1989

Export of prepro-alpha-factor from Escherichia coli.

Yeast prepro-alpha-factor translocates posttranslationally into yeast microsomes in vitro. This process is strongly influenced by the extreme carboxyl-terminal region of the protein. These features contrast with the properties of most eucaryotic proteins which are translocated into the endoplasmic reticulum. We have extended these studies by introducing the gene for the wild-type and several mutant forms of prepro-alpha-factor into Escherichia coli. Prepro-alpha-factor is secreted into the periplasm and processed to pro-alpha-factor. Its translocation across the plasma membrane requires the membrane potential and the secY gene product. Deletion mutant analysis showed that features of the pro-segment were essential for secretion of prepro-alpha-factor in E. coli, while the carboxyl-terminal region, which is required in yeast, is dispensible in E. coli. Neither size nor the presence of a unique topogenic sequence was sufficient to explain the requirement for the pro-segment.

Chromosome Deletion↗
PubMed1989

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.

Amino Acid Isomerases↗
PubMed1989

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.

Adenosine Triphosphatases↗
PubMed1989

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli--SecB, trigger factor and GroEL--have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed 'chaperones'. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation-ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

Adenosine Triphosphatases↗
PubMed1989

Secretion and membrane assembly.

Cytoplasmic proteins undergo rapid and stable folding which buries their apolar segments. In contrast, precursors of secreted and membrane proteins have apolar segments which are recognized by chaperones and membrane receptors to distinguish them from soluble proteins.

Membrane Proteins↗
PubMed1989

Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli.

The ATP-hydrolytic activity of SecA protein is stimulated up to 100-fold by the translocation-competent precursor to outer membrane protein A (pro-OmpA) in conjunction with inner-membrane vesicles bearing active SecY [Lill, R., Cunningham, K., Brundage, L., Ito, K., Oliver, D. & Wickner, W. (1989) EMBO J. 8, 961-966]. This reaction is saturable, with Michaelis-Menten kinetics for an enzyme with two substrates, ATP and pro-OmpA, and is defined as translocation ATPase. Another precursor protein, pre-PhoE, is also a substrate for this translocation ATPase. Neither OmpA nor its synthetic leader peptide are effective substrates for translocation ATPase, suggesting that both domains of the complete precursor are necessary for the reaction. The leader peptide is a potent inhibitor and apparently competes with pro-OmpA for necessary binding sites on translocation ATPase. After a brief preincubation, the activity of translocation ATPase becomes resistant to inhibition by leader peptide, suggesting that the leader peptide is recognized at an early step in the protein translocation pathway. Our enzymological studies show that translocation ATPase recognizes and functionally binds the leader region of precursor proteins.

Amino Acid Sequence↗
PubMed1989

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.

Alkaline Phosphatase↗
PubMed1988

ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle.

We have isolated large amounts of E. coli outer-membrane protein A precursor (proOmpA). Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein. A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form. Comparison of trigger factor's amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene. Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration. Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion. This postribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.

Amino Acid Isomerases↗
PubMed1988

The "trigger factor cycle" includes ribosomes, presecretory proteins, and the plasma membrane.

Trigger factor is a soluble, 63,000 dalton protein of E. coli that stabilizes proOmpA, the precursor form of a major outer-membrane protein, in a conformation competent for in vitro membrane assembly. There is approximately one trigger factor molecule bound to each 70S ribosome isolated from cell extracts in physiological buffers. Trigger factor dissociates from ribosomes in 1.5 M LiCl and reassociates with salt-washed ribosomes in low-salt buffer. Binding is exclusively to the 50S (large) subunit, known to contain the exit domain for nascent polypeptide chains. In addition to its associations with proOmpA and ribosomes, excess trigger factor can compete with the proOmpA-trigger factor complex for a limited number of membrane sites that are essential for translocation of proOmpA. These data suggest a model of trigger factor cycling between the cytoplasm, the ribosome, presecretory proteins, and membrane receptor proteins.

Amino Acid Isomerases↗