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Biomedical subjects

G Schatz

Publications and source records attributed to G Schatz.

At least 109 records · Page 6Linked to original sources
PubMed1991

Protein import into mitochondria: two systems acting in tandem?

Mitochondria can import proteins from the cytoplasm at sites where the two mitochondrial membranes are closely apposed. However, each of these membranes contains a distinct protein translocation channel. Recent evidence suggests that the two types of channel are not permanently coupled, but may dissociate in a reversible manner. This reversible interaction is probably essential for intramitochondrial sorting proteins.

Journal Article↗
PubMed1991

Evaluation of enzyme immunoassay (EIA) as a screening method for hepatitis B markers in an open population.

Commercially available kits for detection of hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) by enzyme immunoassay (EIA) were evaluated in American Samoa during a public health programme to eliminate the transmission of hepatitis B. The first 19,184 serum specimens obtained, representing 68% of the total cooperating population, were initially tested for anti-HBs, and those without detectable antibody were tested for HBsAg. All the antigen-positive serum samples, and a selection of the antigen- and antibody-negative specimens were tested by radioimmunoassay (RIA) for detection of both markers. Compared with the standard tests, the EIA kits for anti-HBs and HBsAg performed well; sensitivity and specificity were 90.3 and 96.0%, respectively, for antibody, and 97.8 and 97.9% respectively for antigen. Substantial disagreement between the EIA and RIA tests for HBsAg was found only for specimens considered weakly reactive by EIA. Few differences were found between three EIA method options for follow-up HBsAg testing of weakly reactive serum specimens; each option contributed about equally to improved test specificity for these 'borderline' specimens. Based on their demonstrated equivalence to the standard RIA tests, we conclude that the EIA kits for anti-HBs and HBsAg detection are suitable for use in hepatitis B control programmes in open populations.

Evaluation Studies as Topic↗
PubMed1990

A yeast mitochondrial outer membrane protein essential for protein import and cell viability.

The gene encoding ISP42, an integral outermembrane protein located at the yeast mitochondrial protein import site was cloned, sequenced and modified. Yeast cells depleted of ISP42 accumulate uncleaved mitochondrial precursor proteins and then die. ISP42 is the first mitochondrial membrane protein shown to be indispensable for protein import and cell viability.

Amino Acid Sequence↗
PubMed1990

The MAS-encoded processing protease of yeast mitochondria. Overproduction and characterization of its two nonidentical subunits.

The amino-terminal presequences of proteins imported from the cytoplasm across the mitochondrial inner membrane are cleaved off by a soluble matrix-localized protease composed of two nonidentical homologous subunits. In the yeast Saccharomyces cerevisiae, these are encoded by the nuclear MAS1 and MAS2 genes. We have now constructed yeast strains in which either one or both of the genomic MAS genes are controlled by a galactose-inducible strong promoter. In these strains, the intramitochondrial concentration of each MAS-encoded subunit as well as of the holo-protease can be varied over a wide range. When overproduced, the MAS1 protein precipitates in the matrix whereas the MAS2 protein remains soluble. The MAS2 protein was obtained at a purity of 98% in milligram amounts. The purified MAS2 subunit exists largely as a soluble 52-kDa monomer. Its cleavage activity is very low and might well reflect the 2% contamination by holoprotease. Activity is restored by adding the solubilized purified MAS1 subunit. Yeast cells depleted of one or both MAS subunits continue to import precursor proteins into mitochondria, but fail to cleave them; eventually the deficient cells stop growing. This growth arrest is partly suppressed on minimal medium or under conditions in which the cells are less dependent on mitochondrial metabolism. Depletion of the MAS1 subunit causes overproduction of the MAS2 subunit.

Cell Nucleus↗
PubMed1990

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.

Cytochromes c1↗
PubMed1990

A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein.

We have probed the environment of a precursor protein stuck in mitochondrial import sites using cleavable bifunctional crosslinking reagents. The stuck precursor was crosslinked to a 70 kd protein which, by immunological techniques, was shown to be a matrix protein. The protein was purified to homogeneity by ATP-Sepharose chromatography and partially sequenced. Fourteen of its 15 N-terminal amino acids were identical to residues 24-38 of the protein encoded by the nuclear gene SSC1, which had been proposed to encode a dnaK-like 70 kd mitochondrial stress protein. Our data imply that this mitochondrial hsp70 is made with a cleavable matrix-targeting sequence composed of 23 residues. The complex containing stuck precursor, mitochondrial hsp70, and ISP42 could be solubilized from mitochondria by the non-ionic detergent Triton X-100 even without crosslinking, suggesting tight association of these three components. As the stuck precursor is arrested at an early stage of translocation, mitochondrial hsp70 may initiate the events that lead to refolding of imported precursors in the matrix space.

Biological Transport↗
PubMed1990

Properties of avian retrovirus particles defective in viral protease.

The structural and enzymatic components of retroviral cores are formed by proteolytic cleavage of precursor polypeptides, mediated by the viral protease (PR). We constructed an active-site mutation, D37I, in the PR of avian leukosis virus. The D37I mutation was introduced into an infectious DNA clone, and quail cell lines expressing the mutant virus were established. These cell lines produce normal amounts of virus particles, the major internal protein components of which are the uncleaved gag and gag-pol precursors. As in other retroviral systems, the protease-defective virions are noninfectious and retain the "immature" type A morphology as determined by thin-section transmission electron microscopy. The virion cores are stable at nonionic detergent concentrations that completely disrupt wild-type cores. Digestion of mutant virions with exogenous PR in the presence of detergent leads to complete and correct cleavage of the gag precursor but incomplete cleavage of the gag-pol precursor. The protease-defective virions encapsidate normal amounts of genomic RNA and tRNA(Trp) that is properly annealed to the primer-binding site, but some of the genomic RNA remains monomeric. Results from UV cross-linking experiments show that the gag polyprotein of mutant virions interacts with viral RNA and that this interaction occurs through the nucleocapsid (NC) domain. However, within mutant virions the interaction of the NC domain with RNA differs from that of mature NC with RNA in wild-type virions. Reverse transcriptase (RT) activity associated with mutant virions is diminished but still detectable. Digestion of the virions with PR leads to a fivefold increase in activity, but this PR-mediated activation of RT is incomplete. Since in vitro cleavage of the gag-pol precursor is also incomplete, we hypothesize that amino acid sequences N terminal to the reverse transcriptase domain inhibit RT activity.

Animals↗
PubMed1989

The mitochondrial targeting function of randomly generated peptide sequences correlates with predicted helical amphiphilicity.

A pool of oligonucleotides encoding a start methionine and nine random amino acids was inserted at the 5'-end of the gene for the yeast cytochrome oxidase subunit IV lacking its own mitochondrial targeting sequence. Approximately one-quarter of the randomly generated sequences targeted subunit IV to its correct intramitochondrial location in vivo. Sequence analysis of 89 randomly generated sequences showed that their efficiencies as mitochondrial targeting signals correlated with the potential to fold into an amphiphilic alpha-helix. Functional targeting sequences were enriched in arginine and isoleucine residues but contained few aspartate, glutamate, and proline residues. Nonfunctional sequences predicted to have significant helical amphiphilicity often had at least one acidic or multiple helix-breaking residues that would be expected to interfere with targeting functioning. These results support the hypothesis that the signal for targeting a protein into the mitochondrial matrix is usually a positively charged amphiphilic helix.

Amino Acid Sequence↗
PubMed1989

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site.

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.

Biological Transport↗
PubMed1989

DNA-protein conjugates can enter mitochondria via the protein import pathway.

Mitochondria import most of their proteins and small molecules from the cytoplasm. There is some tentative evidence that they import some of their RNAs, but it is not known how nucleic acids could enter mitochondria. Here, we show that isolated yeast mitochondria can import a single-stranded or double-stranded 24-base pair piece of DNA whose 5' end is covalently linked to the C-terminus of a mitochondrial precursor protein.

DNA↗