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L L Randall

Publications and source records attributed to L L Randall.

70 records · Page 4Linked to original sources
PubMed1981

Energy is required for maturation of exported proteins in Escherichia coli.

It has been established in numerous cases that proteins which are exported from Escherichia coli are synthesized on membrane-bound polysomes in precursor forms which are proteolytically cleaved to generate the mature species. Here we present evidence that at least one step in the export of proteins requires energy. Energy requirements for processing of the precursors of both the M13 coat protein [Date, T., Zwizinski, C., Ludmerer, S., and Wickner, W. (1980) Proc. Natl Acad. Sci. USA, 77, 827-831; Date, T., Goodman, J. M., and Wickner, W. T. (1980) Proc. Natl Acad. Sci. USA, 77, 4669-4673] and the B subunit of heat-labile enterotoxin [Palva, T., Hirst, T. R., Hardy, S. J. S., Holmgren, J., and Randall, L. L. (1981) J. Bacteriol. in the press] have been demonstrated previously. An energy requirement for the proteolytic processing of an additional five exported proteins is reported here. Studies utilizing an uncA mutant suggest that the form of energy required is proton-motive force. Thus an energized membrane is probably essential for export of most periplasmic and outer membrane proteins.

Adenosine Triphosphate↗
PubMed1981

Processing in vivo of precursor maltose-binding protein in Escherichia coli occurs post-translationally as well as co-translationally.

The mechanism of synthesis of maltose-binding protein (Mr = 38,500), an exported periplasmic protein in Escherichia coli, was investigated in vivo. A precursor to maltose-binding protein (Mr - 41,000), which is identical to the precursor polypeptide synthesized in vitro in a cell-free system, can be detected in vivo indicating that it is not processed to mature size until the polypeptide chain is terminated. The population of incomplete, nascent polypeptide chains of maltose-binding protein was found to contain NH2 termini characteristic of both precursor and mature protein demonstrating that processing occurs co-translationally as well as post-translationally. However, the polypeptide containing the signal sequence must reach a critical size of Mr - 33,000 before any processing takes place.

ATP-Binding Cassette Transporters↗
PubMed1981

Different exported proteins in E. coli show differences in the temporal mode of processing in vivo.

A number of exported proteins in E. coli, both periplasmic proteins and proteins of the outer membrane, were examined to determine when removal of the "signal sequence" occurs in vivo. One protein was processed entirely cotranslationally (amp C beta-lactamase) and one was processed entirely post-translationally (TEM beta-lactamase). The others (maltose-binding protein, arabinose-binding protein, omp A protein, lam B protein and alkaline phosphatase) showed both modes of processing, although the amount of cotranslational processing varied considerably among the individual proteins of this class. When processing occurred cotranslationally, the proteolytic removal of the "signal" was a late event. For four of the proteins studied, processing was initiated only after the polypeptides had been elongated to approximately 80% of their full length.

ATP-Binding Cassette Transporters↗
PubMed1980

Processing of exported proteins in Escherichia coli.

The mechanism of export of protein in E. coli can be summarized in terms of the 'signal hypothesis'. The proteins are synthesized on membrane-bound polyribosomes in the form of precursors, which carry N-terminal extensions of amino acids, the 'signal'. The proteins are vectorially transferred through the membrane during synthesis and the signal sequence is removed to generate the mature protein. The basic principle is established and it is now important to elucidate the molecular mechanism of export. We have attempted to detail the proteolytic removal of the signal. We have shown that the precursors are processed post-translationally, and we have data suggesting that two cleavages may be involved. It appears that processing is not necessary to activate the mature proteins. Why then is the signal removed? Perhaps the answer will shed light on the other yet unanswered questions: what is the energy source for the translocation? how does the cell differentiate between the two classes of exported proteins, those in the periplasm and those in the outer membrane? The next few years should see the resolution of these questions.

ATP-Binding Cassette Transporters↗
PubMed1980

Novel intermediates in the synthesis of maltose-binding protein in Escherichia coli.

Nascent intermediates in the synthesis of maltose-binding protein, a periplasmic protein in Escherichia coli, were demonstrated both in vivo and in vitro. They are likely to result from a drastic reduction in the rate of elongation at specific sites on the mRNA leading to detectable accumulation of distinct species of incomplete polypeptides. In order to reach its final destination in the periplasmic space, maltose-binding protein is transferred across the cytoplasmic membrane as it is elongated. It is possible that variations in the rate of elongation are involved in this export process.

ATP-Binding Cassette Transporters↗
PubMed1978

Precursors of three exported proteins in Escherichia coli.

Arabinose-binding protein, maltose-binding protein, and lambda receptor are synthesized in vitro on membrane-bound polysomes from Escherichia coli. All three proteins are exported from the cytoplasm of E. coli and all three are made in vitro in a form a few thousand daltons larger than the authentic protein. The larger form of arabinose-binding protein is also detected in vivo by pulse labeling. It is concluded that the larger forms of the exported proteins are precursors containing an extra sequence. In contrast to the above, when the intracellular protein elongation factor Tu is synthesized in vitro on free polysomes, it is not detectably larger than the authentic form.

Arabinose↗
PubMed1978

Arrangement of protein I in Escherichia coli outer membrane: cross-linking study.

The arrangement of protein I in the outer membrane of Escherichia coli was investigated by cross-linking whole cells, isolated cell wall, protein-peptidoglycan complexes, and protein I released from peptidoglycan with NaCl. Both cleavable azide cross-linkers and imidoester reagents were used. The data presented suggest that protein I exists in the outer membrane as a trimer.

Azides↗
PubMed1977

Synthesis of exported proteins by membrane-bound polysomes from Escherichia coli.

A membrane-bound fraction of polysomes of Escherichia coli has been isolated after lysis of cells without the use of lysozyme. Protein-synthesis studies in vitro show that membrane-bound and free polysomes are different in the following respects. 1. Membrane-bound polysomes synthesize proteins which are exported from the cell. The products include proteins of the outer membrane and a secreted periplasmic protein, the maltose-binding protein. 2. The major product synthesized by free polysomes is elongation factor Tu, a soluble cytoplasmic protein. 3. The activity of membrane-bound polysomes in vitro is more resistant to puromycin than is the activity of free polysomes. In addition, the mRNA associated with membrane-bound polysomes is more stable than the bulk of cellular mRNA as revealed by studies with rifampicin.

Binding Sites↗
PubMed1975

Analysis of the ribosomes engaged in the synthesis of the outer membrane proteins of Escherichia coli.

The messenger RNAs for the outer membrane proteins in E. coli are more stable than the bulk of the messenger RNA s (Hirashima et al., 1973). Polysomes, enriched in those containing stable mRNAs have been isolated following rifampicin treatment and have been shown to contain quantitatively the same complement of ribosomal protein as normal polysomes. There is one exception: ribosomal protein S1 is present in larger amounts in the polysomes containing stable messengers. However, there are grounds for believing this finding to be an artifact. It is concluded that the differences between outer membrane protein synthesis and bulk protein synthesis are not due to a difference in the ribosomes.

Bacterial Proteins↗
PubMed1975

Quantitation of the loss of the bacteriophage lambda receptor protein from the outer membrane of lipopolysaccharide-deficient strains of Escherichia coli.

The recpetor for the phage lambda, a protein component of the outer membrane, is present at decreased levels in strains of Escherichia coli that are deficient in lipopolysaccharide. Loss of the protein was quantitated both by an assay of the phage receptor function and by an assay of antiserum-blocking ability to detect inactive protein. The loss of protein was correlated with the loss of sugar residues and phosphage from the core region of the lipopolysaccharide. Implications for the importance of ionic interactions in the stabilization of the outer membrane are discussed.

Antibodies, Bacterial↗