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Biomedical subjects

W Wickner

Publications and source records attributed to W Wickner.

At least 109 records · Page 6Linked to original sources
PubMed1985

Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane.

Leader peptidase cleaves the amino-terminal leader sequences of many secreted and membrane proteins. We have examined the function of leader peptidase by constructing an Escherichia coli strain where its synthesis is controlled by the arabinose B promoter. This strain requires arabinose for growth. When the synthesis of leader peptidase is repressed, protein precursors accumulate, including the precursors of M13 coat protein (an inner membrane protein), maltose binding protein (a periplasmic protein), and OmpA protein (an outer membrane protein). These precursors are translocated across the plasma membrane, as judged by their sensitivity to added proteinase K. However, pro-OmpA and pre-maltose binding protein are retained at the outer surface of the inner membrane. Thus, leader peptides anchor translocated pre-proteins to the outer surface of the plasma membrane and must be removed to allow their subsequent release into the periplasm or transit to the outer membrane.

Arabinose↗
PubMed1985

Conserved residues of the leader peptide are essential for cleavage by leader peptidase.

Gene 8 of bacteriophage M13 codes for procoat, the precursor of its major coat protein. Gene 8 has been cloned into a plasmid and mutagenized. We have isolated mutants of this gene in which procoat is synthesized but is not processed to coat protein. We now describe mutants in the leader region of procoat, at positions -6, -3, and -1 with respect to the leader peptidase cleavage site. These positions are quite conserved among the leader peptides of various pre-proteins. Each of these mutant procoats is synthesized at a normal rate and inserts correctly into the plasma membrane, as judged by its accessibility to protease in intact spheroplasts. Procoat accumulates, largely in its transmembrane form, and is not cleaved to coat. In detergent extracts, the mutant procoats are very poor substrates for added leader peptidase. We conclude that these 3 residues are not conserved for insertion across the membrane but are part of an essential recognition site for the leader peptidase.

Bacteriophages↗
PubMed1985

The leader region of pre-maltose binding protein binds amphiphiles. A model for self-assembly in protein export.

Maltose binding protein, like most periplasmic proteins, is resistant to a variety of proteinases. Treatment of pre-maltose binding protein with trypsin, chymotrypsin, or proteinase K removes an amino-terminal domain of the same approximate size as the leader sequence without degrading the mature portion of the protein. In addition, pre-maltose binding protein is as active as mature in binding maltose (Ferenci, T., and Randall, L.L. (1979) J. Biol. Chem. 254, 9979-9981). By these criteria, the precursor and mature proteins are in the same conformation except for the exposed leader sequence on the precursor. We have compared the ability of these proteins to interact with amphipaths, such as detergents. The precursor protein binds to Triton X-100, while the mature protein does not. We propose that the leader domain is responsible for detergent binding. Mutations in the leader region of the precursor which block export in vivo prevent detergent binding in vitro. A mutant with a mild export defect can still bind detergent. This correlation between detergent binding by precursors with related leaders and export efficiency of each precursor suggests that hydrophobic partition of the leader may initiate pre-protein transfer across the membrane.

Amino Acid Sequence↗
PubMed1985

Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage.

The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.

Arabinose↗
PubMed1985

M13 procoat inserts into liposomes in the absence of other membrane proteins.

Procoat, the precursor form of the major coat protein of coliphage M13, assembles into the Escherichia coli inner membrane and is cleaved to mature coat protein by leader peptidase. This assembly process has previously been reconstituted using lipids and purified leader peptidase in a cell-free protein synthesis reaction (Watts, C., Silver, P., and Wickner, W. (1981) Cell 25, 347-353; Ohno-Iwashita, Y., and Wickner, W. (1983) J. Biol. Chem. 258, 1895-1900). We now report that procoat can also cross a liposomal membrane composed of only purified phospholipids; leader peptidase is not needed to catalyze insertion. When procoat is synthesized in vitro in the presence of liposomes with encapsulated chymotrypsin, the procoat inserts spontaneously through the membrane and is degraded. The protease was shown by several criteria to be in the lumen of the liposomes. These results demonstrate that the precursor form of an E. coli integral membrane protein can cross a membrane without the aid of leader peptidase or any other membrane proteins.

Capsid↗
PubMed1985

Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli.

We have examined the effects of thermosensitive mutations in secA and secY (prlA) genes on the export of proteins to the three layers of the Escherichia coli cell surface. After several hours at the nonpermissive temperature, the export of two major outer membrane proteins, lipoprotein and OmpA, is delayed, then essentially blocked, in either a secA or secY strain. These mutations also have a strong effect on the export of several proteins, such as maltose binding protein, to the periplasm, though the export of many periplasmic proteins is not affected. secA and secY block the assembly of leader peptidase, which is made without a leader sequence, into the inner membrane. However, the membrane assembly of M13 coat protein (an inner membrane protein made with an amino-terminal leader sequence) is not affected. Thus, the requirement for sec function for export does not correlate with the presence or absence of leader peptide or with a particular subcellular compartment, but rather is specific to each particular protein.

ATP-Binding Cassette Transporters↗
PubMed1984

Processing of preproteins by liposomes bearing leader peptidase.

Procoat, the precursor form of M13 coat protein, assembles into sealed liposomes bearing only internally oriented leader peptidase and is processed to yield transmembrane coat protein [Ohno-Iwashita, Y., & Wickner, W. (1983) J. Biol. Chem. 258, 1895-1900]. The precursors of maltose-binding protein and of outer membrane protein A (OmpA) are also processed by these liposomes, showing that these preproteins can at least partially insert across a lipid bilayer. The ability to insert into a bilayer may be a general property of preproteins. The cleavage products, mature OmpA and maltose-binding protein, are not sequestered within the liposomes, suggesting that an additional factor(s) is (are) required for complete translocation. Liposomes were also prepared with leader peptidase in a more physiological, membrane-spanning orientation. These liposomes were also active in the cleavage of externally added procoat, pro-OmpA, and pre maltose-binding protein, though the mature OmpA and maltose-binding protein were still not sequestered within the liposomes. Pretreatment of these liposomes with trypsin cleaved near the amino terminus of the leader peptidase, inactivating the enzyme. The function of this amino-terminal domain, on the opposite side of the membrane from the catalytic domain, is unknown.

ATP-Binding Cassette Transporters↗
PubMed1984

Bacterial leader peptidase, a membrane protein without a leader peptide, uses the same export pathway as pre-secretory proteins.

Leader peptidase typifies a group of proteins of the plasma membrane of E. coli which span the membrane and are synthesized without a cleaved amino-terminal leader (signal) sequence. The membrane assembly properties of these proteins have not been previously reported. We find that the membrane electrochemical potential is necessary for the insertion of a large domain of leader peptidase across the membrane. In the absence of potential, the peptidase accumulates inside the cell in tight association with the plasma membrane. Upon restoration of the potential, accumulated peptidase inserts across the membrane, indicating that this insertion is not mechanistically coupled to polypeptide chain growth. The normal, trans-bilayer peptidase and that which accumulates in the absence of potential have different conformations, as shown by the relative resistance of the trans-bilayer enzyme to digestion by trypsin or chymotrypsin in cell lysates. Membrane insertion is accompanied by this conformational change. This assembly reaction has several features predicted by the hypothesis of membrane-triggered folding.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1983

Sequence of the leader peptidase gene of Escherichia coli and the orientation of leader peptidase in the bacterial envelope.

The nucleotide sequence of the leader peptidase structural gene from Escherichia coli has been determined. The gene codes for a protein of 323 amino acid residues with a calculated Mr = 35,994, in agreement with the apparent molecular weight of leader peptidase (37,000) determined from gel electrophoresis in sodium dodecyl sulfate. In addition, the amino acid composition predicted from the DNA sequence matches that of the purified enzyme. Leader peptidase synthesized in a cell-free transcription-translation system and isolated from cell membranes have identical apparent molecular weights on sodium dodecyl sulfate-polyacrylamide gels. The presence of methionine and cysteine in the NH2-terminal tryptic peptide shows that no more than three amino acid residues have been removed from the primary translation product predicted from the DNA sequence. These results indicate that leader peptidase is synthesized and assembled into the membrane without proteolytic removal of a leader peptide. Leader peptidase is shown to be largely found in the inner membrane of overproducing strains. The majority of the polypeptide chain is exposed on the outer surface of the inner membrane. It is anchored by a membrane-spanning segment near the NH2 terminus. This orientation agrees with the functional orientation of leader peptidase observed in artificial membrane vesicles.

Amino Acid Sequence↗
PubMed1983

Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli.

OmpA is a major protein of the outer membrane of Escherichia coli. It is made as a larger precursor, pro-OmpA, which requires a membrane potential for processing. We now show that pro-OmpA accumulates in the cytoplasm of cells treated with carbonyl cyanide m-chlorophenylhydrazone, an uncouple which lowers the membrane potential. Upon restoration of the potential, this pro-OmpA is secreted, processed, and assembled into the outer membrane. Pro-OmpA made in vitro is also recovered with the postribosomal supernatant. It is efficiently processed to OmpA by liposomes which have bacterial leader peptidase that is exclusively internally oriented. These experiments show that: (i) the insertion of pro-OmpA into the plasma membrane is not coupled to its synthesis; (ii) insertion is promoted by the transmembrane electrochemical potential; (iii) pro-OmpA can cross a bilayer spontaneously; and (iv) pro-OmpA is processed by the same leader peptidase which converts M13 procoat to coat.

Bacterial Outer Membrane Proteins↗
PubMed1983

Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase.

The leader peptidase of Escherichia coli cleaves a 23-residue leader sequence from M13 procoat to yield mature coat protein in virus-infected cells. We have reconstituted pure leader peptidase into vesicles of E. coli lipids and found that these liposomes are active in the conversion of procoat to coat. Trypsin removes all but 10% of the leader peptidase, yet the vesicles retain nearly full capacity to convert procoat to coat, suggesting that only procoat which inserts across the liposomal membrane is a substrate for leader peptidase. This is confirmed by the finding that over 70% of the coat protein produced by these liposomes spans the membrane. The rate at which leader peptidase inside protease-treated liposomes cleaves externally added procoat is comparable to the rate of procoat cleavage by the same amount of leader peptidase in detergent micelles. Thus, procoat can rapidly integrate across a liposomal membrane and be cleaved to coat protein. These findings confirm the central part of the membrane trigger hypothesis that certain proteins (such as procoat) can cross a bilayer without the aid of a proteinaceous pore or transport system.

Antigen-Antibody Complex↗
PubMed1983

M13 procoat and a pre-immunoglobulin share processing specificity but use different membrane receptor mechanisms.

Bacteriophage M13 procoat is accurately processed to transmembrane coat protein by salt-washed or N-ethylmaleimide-treated rough microsomes from dog pancreas. These treatments inhibit the processing of eukaryotic secreted protein precursors. M13 procoat can assemble into dog pancreas microsomes post-translationally. Thus, the microsomal proteins needed for assembly may be determined by the nature of the precursor protein itself. These results, and our finding that the mouse IgG kappa chain fragment precursor is processed by Escherichia coli leader peptidase, also suggest that the cleavage specificity of leader (signal) peptidases and the properties of preproteins that render them suitable for cleavage have been conserved during evolution.

Animals↗
PubMed1983

Genetic mapping of the Escherichia coli leader (signal) peptidase gene (lep): a new approach for determining the map position of a cloned gene.

The gene for leader peptidase, termed lep, was mapped to the region between purI and nadB at min 54 to 55 on the Escherichia coli chromosome. Mapping involved (i) cloning the gene into the plasmid pBR322, (ii) transforming the plasmid into a polA strain where it cannot replicate autonomously, (iii) selecting by ampicillin resistance the rare cell in which the plasmid had recombined into the chromosome, and (iv) mapping the chromosomal site of drug resistance (and thus plasmid integration) by Hfr matings and P1 transduction. The map position was confirmed by an assay of the enzyme content of cells bearing an F' factor which covered that region of the chromosome.

Ampicillin↗
PubMed1982

The isolation of homogeneous leader peptidase from a strain of Escherichia coli which overproduces the enzyme.

Leader (signal) peptidases cleave the NH2-terminal leader sequences of newly synthesized secreted and membrane proteins during, or shortly after, they insert across the membrane. We have constructed a plasmid, pPS9, in which the structural gene for Escherichia coli leader peptidase is under transcriptional control of a lambda promoter, PR. pPS9 also codes for a temperature-sensitive lambda repressor, causing repression of expression of the plasmid leader peptidase gene at 30 degrees C and rapid synthesis of this enzyme at 42 degrees C. Under appropriate growth regimens, leader peptidase is overproduced 75-fold. Leader peptidase from this strain has been purified to homogeneity. It consists of a 37,000-dalton polypeptide which co-migrates with enzyme activity on DEAE-cellulose and chromatofocusing chromatography. Antibody to this leader peptidase is described.

DNA Restriction Enzymes↗