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M N Hall

Publications and source records attributed to M N Hall.

At least 73 records · Page 4Linked to original sources
PubMed1987

Signal sequence mutations that alter coupling of secretion and translation of an Escherichia coli outer membrane protein.

The lamB701-708 signal sequence mutation reduces expression of LamB, an outer membrane protein of Escherichia coli. To investigate the possibility that synthesis and export of LamB are coupled, as suggested by the expression defect of the lamB701-708 mutation, we isolated intragenic suppressors of the lamB701-708 mutation. The expression defect imposed by the lamB701-708 mutation is suppressed by an export-defective signal sequence mutation, suggesting that translation and export are coupled. The additional observation that not all export-defective signal sequence mutations suppressed the lamB701-708 expression defect suggests that translational arrest can be uncoupled from export.

Amino Acid Sequence↗
PubMed1984

Targeting of E. coli beta-galactosidase to the nucleus in yeast.

In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.

Base Sequence↗
PubMed1983

Inactivation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by a plasma acetylhydrolase: higher activities in hypertensive rats.

We have partially characterized the properties of a specific acetylhydrolase in plasma from spontaneous hypertensive rats. This enzyme inactivates 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (a lipid involved in platelet aggregating, hypotensive, and allergic responses) by removal of the acetate group. The extent of acetate hydrolysis was linear with both time and protein concentration, and the enzyme had an apparent Km of 2.5 microM and a Vmax of 2.6 nmol/min/mg protein. As with an intracellular acetylhydrolase previously characterized by us, the plasma activity was not affected by addition of phosphatidylcholine, EDTA, or Ca2+. However, in contrast to the acetylhydrolase activity in the rat kidney soluble fraction, the plasma activity was associated with a higher molecular weight protein resolved on a Sepharose 6B column and the plasma acetylhydrolase was not inhibited by treatment with trypsin, pronase, or subtilisin. We also compared the acetylhydrolase activity in plasma of age-matched spontaneous hypertensive rats and their normotensive controls, and found approximately 20% higher levels of activity in plasma from the hypertensive animals (P less than 0.01).

Animals↗
PubMed1983

Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine.

Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12. Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively. A third locus, ompB, is defined by mutations that map at 74 minutes. The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression. One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters. Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype. The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner. Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins. In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e. OmpF- OmpC+. Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations. One of these classes exhibits an OmpF+ OmpC- phenotype. The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions. Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor. These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.

Bacterial Outer Membrane Proteins↗
PubMed1983

Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli.

We describe a lesion, lamB701-708, affecting the hydrophilic portion of the lambda receptor signal sequence. The C to A transversion of the sixth codon of the signal sequence changes a positively charged arginine to a neutral serine. The phenotype conferred by this alteration is unique among previously described signal sequence mutations. The results suggest an essential role for the charged amino acids of the hydrophilic segment in the initial interaction between a nascent secreted protein and a membrane export site. The results further suggest that synthesis of lambda receptor is coupled to its export.

Artificial Gene Fusion↗
PubMed1982

Reconsidering the early steps of protein secretion.

We have reviewed a unique mutation affecting the hydrophilic segment of the lambda-receptor signal sequence. Considering the implications of this mutation, we have proposed a model depicting early steps of protein secretion. The salient, novel features of this model are as follows. 1) The model explicitly states that the LamB protein must initiate the export process to be synthesized. Commitment to synthesis occur subsequent to an interaction between the hydrophilic segment of the signal sequence and a membrane export site, the initial interaction between the nascent secreted protein and the membrane. Implicit in this concept is obligatory co-translational secretion of lambda receptor. 2) We suggest the existence of a "stop translation" sequence. The role of this sequence is to halt translation in order to allow sufficient time for the hydrophilic portion of the signal sequence to initiate export by interacting with a membrane receptor. 3) We suggest that at least part of the "stop translation" sequence is located down-stream from the signal sequence, after residue 15 of the mature LamB protein. 4) We further speculate, albeit in the absence of direct evidence, that the hydrophobic portion of the signal sequence may also be part of the "stop translation" sequence.

Amino Acid Sequence↗
PubMed1981

Identification of OmpR: a positive regulatory protein controlling expression of the major outer membrane matrix porin proteins of Escherichia coli K-12.

We report here on the cloning of a gene located within the ompB locus of E. coli K-12. This gene, designated ompR, resides on a 10.9-kilobase EcoRI fragment that we cloned, using a lambda vector and in vitro packaging techniques. By subcloning portions of this fragment into the high-copy-number plasmid pBR322, we have isolated the ompR gene on a 1.4-kilobase EcoRI-AvaI fragment. This fragment has been characterized physically and will facilitate a more detailed study of the role and mechanism of porin regulation by the ompB locus.

Bacterial Proteins↗
PubMed1980

Genetic studies on mechanisms of protein localization in Escherichia coli K-12.

In the last few years, several laboratories have demonstrated that many proteins (both from eukaryotic and prokaryotic organisms) that are destined to be localized in noncytoplasmic locations initially are synthesized as a precursor with a 15-30 amino acid extension at the NH2-terminal end of the molecule. This extra peptide has been termed the signal sequence, and it has been proposed that this signal plays a role in the localization of the extracytoplasmic protein. We are studying the process by which proteins are exported to the envelope region of Escherichia coli. Our work deals primarily with the outer membrane proteins, lambda receptor, the product of the lamB gene, and the major outer membrane (porin) proteins 1a and 1b, products of the ompF and ompC genes. Using techniques of gene fusion, we have demonstrated that information specifying the cellular location of the lambda receptor is contained within the lamB gene. Furthermore, we have shown that this information is capable of directing even a normally cytoplasmic protein, beta-galactosidase, to the outer membrane. Some of this information is contained within the signal sequence. Mutations that alter this sequence prevent export of the lambda receptor protein. Again using techniques of gene fusion, we have shown that the signal sequence alone is not sufficient to cause export of beta-galactosidase from the cytoplasm. Other information within the lamB gene is required. Selection procedures have been developed to isolate mutations that exhibit a general alteration in the export process. Genetic analysis of these mutations has provided evidence for the involvement of the ribosome in the process of protein localization. The structural genes for the porin proteins, 1a and 1b, are regulated at the transcriptional level by the ompB locus. This has permitted us to extend our studies on outer membrane protein localization to protein 1. With this genetic system, it should be possible to determine if E coli employs more than a single mechanism for the export of proteins to the outer membrane.

Bacterial Proteins↗
PubMed1980

A mechanism of protein localization: the signal hypothesis and bacteria.

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.

Bacterial Proteins↗
PubMed1979

Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b.

Eleven independent insertion mutations were isolated that prevented expression of major outer membrane protein 1b. Seven of the mutations were Mucts insertions located at ombP. These ompB::Mucts strains fell into two phenotypic classes with regard to expression of proteins 1a and 1b. The remaining four mutants were comprised of one Tn5 and three Mucts insertions mapping at par. The Mucts insertions at par were used to construct fusions of the lac operon to the par promoter. Expression of beta-galactosidase in these fusion strains reflected known regulatory properties of protein 1b. When an ompB allele was introduced into the par-lac fusion strains, beta-galactosidase activity was reduced 14- to 31-fold. Transcriptional regulation of the par gene and the existence of two functions at ompB are discussed. The results suggest that par is the structural gene for protein 1b and that an ompB gene product is a diffusible, positive regulatory element controlling expression of par.

Bacterial Proteins↗