Biomedical subjects

E Schiebel

Publications and source records attributed to E Schiebel.

42 records · Page 3Linked to original sources

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↗

PubMed1989

Subcellular location and unique secretion of the hemolysin of Serratia marcescens.

It is shown that Serratia marcescens exports a hemolysin to the cell surface and secretes it to the extracellular space. Escherichia coli containing the cloned hemolysin genes shlA and shlB exported and secreted the S. marcescens hemolysin. A nonhemolytic secretion-incompetent precursor of the hemolysin, designated ShlA*, was synthesized in a shlB deletion mutant and accumulated in the periplasmic space of E. coli. Immunogold-labeled ultrathin sections revealed ShlA* bound to the outer face of the cytoplasmic membrane and to the inner face of the outer membrane. A number of mutants carrying 3' deletions in the shlA gene secreted truncated polypeptides, the smallest of which contained only 261 of the 1578 amino acids of the mature ShlA hemolysin, showing that the information for export to the cell surface of E. coli and secretion into the culture medium is located in the NH2-terminal segment of the hemolysin. We propose a secretion pathway in which ShlA and ShlB are exported across the cytoplasmic membrane via a signal sequence-dependent mechanism. ShlB is integrated into the outer membrane. ShlA is translocated across the outer membrane with the help of ShlB. During the latter export process or at the cell surface, ShlA acquires the hemolytically active conformation and is released to the extracellular space. The hemolysin secretion pathway appears to be different from any other secretion system hitherto reported and involves only a single specific export protein.

Escherichia coli↗

PubMed1989

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes.

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.

Absorption↗

PubMed1988

Molecular characterization of the hemolysin determinant of Serratia marcescens.

The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.

Amino Acid Sequence↗

PubMed1987

Identification of the Serratia marcescens hemolysin determinant by cloning into Escherichia coli.

A cosmid bank of Serratia marcescens was established from which DNA fragments were cloned into the plasmid pBR322, which conferred the chromosomally encoded hemolytic activity to Escherichia coli K-12. By transposon mutagenesis with Tn1000 and Tn5 IS50L::phoA (TnphoA), the coding region was assigned to a DNA fragment, designated hly, comprising approximately 7 kilobases. Two proteins with molecular weights of 61,000 (61K protein) and 160,000 (160K protein) were expressed by the pBR322 derivatives and by a plasmid which contained the hly genes under the control of a phage T7 promoter and the T7 RNA polymerase. When strongly overexpressed the 160K protein was released by E. coli cells into the extracellular medium concomitant with hemolytic activity. The genes encoding the 61K and the 160K proteins were transcribed in the same direction. Mutants expressing a 160K protein truncated at the carboxy-terminal end were partially hemolytic. Hemolysis was progressively inhibited by saccharides with increasing molecular weights from maltotriose (Mr 504) to maltoheptaose (Mr 1,152) and was totally abolished by dextran 4 (Mr 4,000). This result and the observed influx of [14C]sucrose into erythrocytes in the presence of hemolytic E. coli transformants under osmotically protective conditions suggest the formation of defined transmembrane channels by the hemolysin.

Bacterial Proteins↗

PubMedDate not supplied

Microtubule organization by the budding yeast spindle pole body.

In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.

Spindle Apparatus↗