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Biomedical subjects

W Wickner

Publications and source records attributed to W Wickner.

At least 127 records · Page 7Linked to original sources
PubMed1982

The biosynthesis of membrane-bound M13 coat protein. Energetics and assembly intermediates.

The major coat protein of bacteriophage M13 spans the plasma membrane of infected cells prior to its assembly into extruding virus. It is initially made as a precursor, termed procoat, with a 23-residue leader sequence at its NH2 terminus. Procoat is found bound to the inner surface of the plasma membrane. The electrical potential of the cell membrane is required for procoat insertion and conversion to coat protein, although the order of these events has been unknown. We now report studies of the conversion of a mutant procoat (procoat-R6) from the virus M13am8H1R6 to mutant coat (coat R6). The behavior of procoat-R6 differs from that of the wild type procoat in three respects. (i) Pulse-labeled procoat-R6 is largely found inserted across the cell membrane. This suggests that the active site of leader peptidase is on the periplasmic membrane face and that insertion normally precedes processing for wild type procoat as well. (ii) Despite the greater abundance of inserted procoat-R6 in M13am8H1R6-infected cells than inserted procoat in wild type infections, procoat-R6 is processed to coat-R6 more slowly than procoat is converted to coat. (iii) The membrane insertion and proteolytic processing of procoat-R6 are almost completely insensitive to uncouplers. We present a working model for the energetics and assembly intermediates of coat protein biosynthesis.

Bacterial Proteins↗
PubMed1982

The purification of M13 procoat, a membrane protein precursor.

Many membrane proteins and most secreted proteins are initially made as precursors with an N-terminal leader sequence. We now report the isolation of M13 procoat, the precursor of the membrane-bound form of M13 coat protein. There are 40 000 copies of M13 procoat protein/cell during M13 amber 7 virus infection. Purified procoat is quantitatively cleaved by isolated leader peptidase to yield mature-length coat protein. Rabbit antibodies to M13 procoat will precipitate procoat but not coat, suggesting that the antibody molecules are specifically recognizing the leader sequence or the conformation which it induces in the whole procoat molecule.

Antigen-Antibody Complex↗
PubMed1981

Leader peptidase is found in both the inner and outer membranes of Escherichia coli.

Many membrane proteins are synthesized as transient precursors with an NH2-terminal leader (or signal) peptide. During insertion of these proteins into the membrane, leader peptides are removed by leader peptidase. One such enzyme has been detected in detergent extracts of Escherichia coli membranes and extensively purified using as an assay the removal of the leader sequence of procoat, the precursor of the major coat protein of bacteriophage M13. We now report that this leader peptidase is found in equal abundance in the inner and outer membranes of E. coli. Enzyme from each membrane accurately cleaves procoat to mature M13 coat protein. The salt, pH, and Mg2+ optima and inhibitor sensitivities of enzyme from each membrane are identical. Furthermore, the activities are indistinguishable upon ion exchange chromatography and nondenaturing gel electrophoresis. Finally, a strain of E. coli with a plasmid which causes overproduction of leader peptidase has elevated levels of enzyme in both the inner and outer membranes. Leader peptidase is the only known enzyme which is found in both inner and outer membrane fractions of E. coli; this may reflect its role in membrane biogenesis.

Cell Membrane↗
PubMed1981

Membrane assembly from purified components. I. Isolated M13 procoat does not require ribosomes or soluble proteins for processing by membranes.

The coat protein of coliphage M13 is an integral protein of the host-cell cytoplasmic membrane prior to its assembly into virions. It is initially synthesized as procoat, a soluble precursor with a 23 amino acid leader sequence at its amino terminus. 35S-labeled procoat accumulates during an in vitro translation reaction that contains 35S-methionine and RNA from M13-infected cells. Radiochemically pure procoat has been isolated from in vitro translation reactions by extraction into an organic solvent and gel filtration through Sephadex LH-60. Radiochemically pure procoat can be used as substrate in rapid and quantitative assays for leader peptidase and for leader peptide hydrolase, an enzyme that degrades the leader peptide after its release from procoat. Procoat solubility, digestion by leader peptidase and processing by membranes are affected by the presence of Mg2+ ion. Isolated procoat is soluble in water at low ionic strength and mildly alkaline pH as well as in detergent solutions. It is cleaved to coat protein by purified E. coli leader peptidase and by inverted E. coli inner-membrane vesicles. These properties of the purified procoat mirror those of the procoat in crude extracts. This suggests that there are no other soluble components that are necessary for the assembly of procoat into the membrane and its conversion to coat; specifically, it provides powerful evidence that protein synthesis is not involved.

Cell Membrane↗
PubMed1981

Membrane assembly from purified components. II. Assembly of M13 procoat into liposomes reconstituted with purified leader peptidase.

The major coat protein of coliphage M13 is an integral protein of the E. coli plasma membrane prior to its assembly into new virus particles. It is generated from its precursor, procoat, by a membrane-bound leader peptidase. We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E. coli phospholipids. These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis. Both the crude and the purified substrates were converted post-translationally to coat protein. A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium. These results strongly suggest that highly purified leader peptidase from E. coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein.

Coliphages↗
PubMed1981

Membrane assembly: posttranslational insertion of M13 procoat protein into E. coli membranes and its proteolytic conversion to coat protein in vitro.

The major coat protein (gene 8 product) of bacteriophage M13 is an integral membrane protein during infection of host cells. It is synthesized as a larger precursor (procoat) with a leader sequence of 23 amino acids at its amino terminus. In vivo studies have shown that procoat only inserts into the host-cell plasma membrane after its synthesis is completed. We now demonstrate that procoat can post-translationally insert into inverted cytoplasmic membrane vesicles from E. coli and can be processed proteolytically to yield coat protein. Procoat changes from an assembly-competent substrate to an incompetent (denatured) form within minutes after its synthesis; much of the procoat that accumulates during an hour of in vitro synthesis is therefore denatured. These studies emphasize the importance of stringent criteria for the demonstration of obligate cotranslational assembly.

Cell Membrane↗
PubMed1981

Isolation of the Escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo.

The only covalent modifications known to accompany protein insertion into membranes or protein secretion are glycosylation and the proteolytic removal of an NH2-terminal leader (signal) sequence. This latter reaction is catalyzed by leader peptidase, a constitutive, membrane-bound proteinase. We now report the identification of a plasmid-bearing strain of Escherichia coli that overproduces leader peptidase 4- to 6-fold. This strain grows normally and shows an unaltered polypeptide composition of inner ad outer membranes. The leader peptidase gene has been subcloned and transferred from this plasmid to the multicopy plasmid pBR322, yielding a new plasmid (pTD101). Strains transformed by pTD101 have a 30-fold increase in leader peptidase. We have studied the effect of leader peptidase overproduction on the insertion of newlymade M13 phage coat protein into the plasma membrane of infected cells. The overproducer strain, when infected by M13 phage, shows a dramatic acceleration in the conversion of procoat (a cytoplasmic precursor form) to coat (an integral, transmembrane protein). Thus the leader peptidase that converts M13 procoat to coat in vitro can catalyze this reaction in vivo as well.

Coliphages↗
PubMed1980

Assembly of proteins into membranes.

Two pathways for protein assembly into biological membranes have been proposed. The "signal hypothesis" emphasizes the role of specific membrane proteins in binding the growing polypeptide and conducting it into the bilayer during its synthesis. The "membrane-triggered folding" hypothesis emphasizes self-assembly and the role of changing protein conformation during transfer from an aqueous compartment into a membrane. These ideas provide a framework for reviewing recent data on the biogenesis of membrane proteins.

Biological Transport↗
PubMed1980

Purification and characterization of leader (signal) peptidase from Escherichia coli.

Many membrane proteins and secreted proteins are synthesized in precursor form with 15 to 30 additional NH2-terminal residues. These "leader peptides" (pre-pieces, signal peptides) are removed as these proteins cross or insert into cellular membranes. "Leader peptidase" activities which catalyze this cleavage have been detected in crude extracts and found to be dependent on membrane fractions. We now describe a 6,000-fold purification of a leader peptidase from the membranes of uninfected Escherichia coli. This leader peptidase was assayed by its ability to cleave the 23-residue leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Immunoprecipitation and amino acid sequencing showed that this enzyme cleaved procoat to produce authentic coat protein. No factors other than the leader peptidase were found to be required for the conversion of procoat protein to coat protein.

Antibodies↗
PubMed1980

Melittin forms crystals which are suitable for high resolution X-ray structural analysis and which reveal a molecular 2-fold axis of symmetry.

Melittin is the principal protein component of bee venom and is believed to function as a lytic agent. In aqueous salt solution, it is a tetramer of identical peptides, each with 26 amino acid residues. Although its amino acid composition is unusually nonpolar, and although it is believed to integrate into membranes while lysing cells, melittin is water-soluble at neutral pH. Two crystal forms have been grown from solutions containing ammonium sulfate and sodium formate, and their x-ray diffraction patterns indicate that the melittin tetramer contains at least one 2-fold axis of rotation. Both crystal forms are suitable for high resolution x-ray structural studies. Moreover, both crystals bind several heavy atoms as judged by changes in buoyancy, so that phase determination by the method of isomorphous replacement is possible. Crystallized melittin retains its lytic activity even under the conditions of crystallization (about 70% saturated ammonium sulfate).

Animals↗
PubMed1980

Mechanisms of membrane assembly: effects of energy poisons on the conversion of soluble M13 coliphage procoat to membrane-bound coat protein.

The coat protein (gene 8 product) of coliphage M13 spans the host cell plasma membrane prior to its assembly into extruding virions. It is made as a soluble precursor, termed procoat, with an extra 23 NH2-terminal amino acid residues. We have examined the effect of metabolic poisons on the assembly of procoat into the plasma membrane and its proteolytic conversion to coat protein. Protein synthesis and proline uptake were measured to assess the effect of each poison on cellular high-energy phosphate and on the transmembrane protonmotive force, respectively. Arsenate, which abolished protein synthesis but did not affect proline uptake, had no measurable effect on the conversion of procoat to coat protein. In contrast, the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) blocked conversion of procoat to coat protein. Neither CCCP nor arsenate inhibited the ability of a detergent-solubilized and highly purified preparation of leader peptidase to convert procoat to coat protein in the presence of detergents. The procoat that accumulated in the presence of CCCP was membrane bound. A spontaneous mutant that grows in the presence of CCCP showed (i) CCCP-resistant proline uptake in whole cells, (ii) CCCP-resistant uptake in inner membrane vesicles, and (iii) CCCP-resistant conversion of procoat protein to coat protein. These data suggest that an electrochemical gradient is at least indirectly necessary for the proper assembly of procoat into the cellular membrane.

Arsenates↗
PubMed1979

Interactions of melittin, a preprotein model, with detergents.

Bee venom melittin is a water-soluble tetramer of identical polypeptide chains. Each chain has 26 residues. The 20 N-terminal residues are hydrophobic and the 6 C-terminal residues are basic. Melittin has been shown to integrate into natural and synthetic membranes and to lyse a wide variety of cells. To understand how a water-soluble protein can spontaneously partition into a membrane, we have studied the interaction of melittin with micelles of deoxycholate (DOC), Brij 58, and sodium dodecyl sulfate (NaDodSO4). Circular dichroism spectra showed that NaDodSO4, an ionic detergent, and Brij 58, a nonionic detergent, caused similar major changes in the protein's conformation. Gel filtration studies revealed that melittin forms mixed micelles with either Brij or DOC. The melittin-DOC mixed micelles have 2 mol of DOC per mol of melittin. Cross-linking studies with dimethyl suberimidate confirmed that the protein is a tetramer and showed that it becomes monomeric either in mixed micelles with Brij or DOC or in butanol. Despite this major structural change of melittin in the presence of an amphiphile, the covalently cross-linked form is as active in human erythrocyte lysis as the native protein.

Bee Venoms↗
PubMed1979

Translational and post-translational cleavage of M13 procoat protein: extracts of both the cytoplasmic and outer membranes of Escherichia coli contain leader peptidase activity.

The coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane at all stages of the infectious cycle. Both in in vivo and DNA-directed in vitro synthesis, it is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed procoat. We now report that leader peptidase, and activity which removes the leader peptide and converts procoat to coat, is found in both the inner (cytoplasmic) and outer membrane of Escherichia coli. However, only cytoplasmic membranes will catalyze cleavage of procoat in the absence of detergent. Leader peptidase will cleave procoat either during translation or after protein synthesis is complete.

Cell Membrane↗
PubMed1979

Soluble precursor of an integral membrane protein: synthesis of procoat protein in Escherichia coli infected with bacteriophage M13.

Prior to virus assembly, the major coat protein of coliphage M13 is an integral protein of the host cytoplasmic membrane. Coat protein synthesized in vitro is initially made with an NH2-terminal "leader peptide" of 23 amino acids and is termed "procoat." We now report that procoat is a biosynthetic precursor of coat protein in vivo. Conversion of procoat to coat occurs within 30 sec in cells infected with wild-type virus. This proteolytic processing is delayed in cells infected by M13 mutants (in genes 1, 5, or 7) that are defective in virus assembly. Pulse--chase experiments in combination with subcellular fractionation show that procoat is synthesized in a soluble form in the cytoplasm and is then incorporated into the cytoplasmic membrane, where it is converted to coat protein. This finding is supported by the observation that procoat is synthesized exclusively by polysomes that are not membrane bound. These results are interpreted in terms of the "membrane-triggered folding" hypothesis of membrane protein assembly.

Bacterial Proteins↗
PubMed1978

Synthesis of phage M13 coat protein and its assembly into membranes in vitro.

The coat protein (gene 8 product) of coliphage M1O is an integral protein of the host cell membrane at all stages of virus infection. This protein, when made in a cell-free reaction, has been shown by others to have an additional NH2-terminal peptide region and is referred to as "procoat." It is initially not membrane-bound but, upon exposure to Escherichia coli membrane vesicles or to liposomes prepared from E. coli lipids, it assembles into the bilayer in an integral fashion. Much of this protein is shown to be exposed on the inner surface of the liposome. We suggest that refolding of procoat as it encounters the bilayer is sufficient to transport large segments of the peptide chain through the apolar hydrocarbon core.

Cell-Free System↗