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Biomedical subjects

G Schatz

Publications and source records attributed to G Schatz.

At least 127 records · Page 7Linked to original sources
PubMed1989

Binding of a tightly folded artificial mitochondrial precursor protein to the mitochondrial outer membrane involves a lipid-mediated conformational change.

An artificial mitochondrial precursor protein (the presequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase) binds to isolated yeast mitochondrial outer membranes and to liposomes whose phospholipid composition resembles that of outer membranes. In both cases, binding is strongly inhibited by low temperature or methotrexate (which stabilizes the dihydrofolate reductase moiety) and partly inhibited by adriamycin (which binds to acidic phospholipids). Binding is accompanied by partial unfolding of the protein. Binding of the urea-denatured fusion protein to outer membranes or liposomes is insensitive to low temperature, methotrexate, or adriamycin. These results, and those reported in the accompanying paper (Eilers, M., Endo, T., and Schatz, G. (1989) J. Biol. Chem. 264, 2945-2950) suggest that import of this fusion protein into isolated mitochondria involves at least partial unfolding by acidic phospholipids on the mitochondrial surface.

Doxorubicin↗
PubMed1989

Adriamycin, a drug interacting with acidic phospholipids, blocks import of precursor proteins by isolated yeast mitochondria.

Acidic phospholipids such as cardiolipin partially unfold an artificial precursor protein which consists of a mitochondrial presequence fused to mouse dihydrofolate reductase (Endo, T., and Schatz, G. (1988) EMBO J. 7, 1153-1158). We now show that import of this precursor protein into isolated yeast mitochondria is blocked by adriamycin, a drug binding to cardiolipin and other acidic phospholipids. This inhibition is lessened if the precursor's dihydrofolate reductase moiety is labilized by point mutations; inhibition is abolished altogether if the "wild-type" precursor is presented to mitochondria in a urea-denatured state. These and other observations suggest that adriamycin interferes with the generation of a translocation-competent, loose structure of the precursor protein. They imply that acidic phospholipids such as cardiolipin participate, directly or indirectly, in the translocation of this fusion protein into isolated mitochondria.

Doxorubicin↗
PubMed1989

Translocation of proteins across the mitochondrial inner membrane, but not into the outer membrane, requires nucleoside triphosphates in the matrix.

Work in several laboratories has established that import of proteins across the mitochondrial inner membrane requires an electrochemical potential across that membrane and cleavage of nucleoside triphosphate. We now show that nucleoside triphosphate must be present inside the inner membrane. In contrast, the potential-independent insertion of porin into the outer membrane requires ATP only outside the inner membrane.

Adenosine Triphosphate↗
PubMed1989

Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane.

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.

Antibodies↗
PubMed1989

Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins.

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.

Adenosine Triphosphate↗
PubMed1989

Interaction between mitochondria and the nucleus.

The interaction between the mitochondrial and the nuclear genome is in part mediated by proteins (and possibly also RNAs) which are encoded in the nucleus and imported into mitochondria. We are beginning to understand how proteins can penetrate across both mitochondrial membranes and how some of these proteins can regulate the expression of specific mitochondrial genes.

Amino Acid Sequence↗
PubMed1988

Import of proteins into yeast mitochondria: the purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes.

We have purified the metalloprotease which is localized in the soluble matrix space of Saccharomyces cerevisiae mitochondria and cleaves the amino-terminal matrix-targeting sequences from imported mitochondrial precursor proteins. The enzyme consists of two loosely associated non-identical subunits of mol. wt 48,000 and 51,000, respectively. Attempts to separate the two subunits from each other caused loss of activity. The smaller subunit had been identified as the product of the nuclear MAS1 gene (Witte et al., 1988). The larger subunit is now identified as the product of the nuclear MAS2 gene.

Alcohol Dehydrogenase↗
PubMed1988

Mutations restoring import of a yeast mitochondrial protein with a nonfunctional presequence.

An internal deletion in the presequence of the precursor to yeast cytochrome oxidase subunit IV blocks import of the protein into mitochondria. We have identified two mechanisms by which yeast cells can suppress the defect of the plasmid-borne defective gene. One mechanism creates a new functional presequence by point mutations, or local DNA rearrangements, within the open reading frame or its 5'-untranslated region. The second mechanism compensates for the defective presequence by recessive mutations in single nuclear genes. The plasmid-linked mutations may mimic the mechanism(s) by which mitochondrial presequences arose during evolution whereas the chromosomal mutations may help to identify components of the mitochondrial import machinery.

Amino Acid Sequence↗
PubMed1988

Protein translocation across membranes.

Many newly synthesized proteins must be translocated across a membrane to reach their final destinations. Translocation requires a signal on the protein itself, a loose conformation of the protein, energy, and receptor-like components in the cytosol and on the target membrane.

Animals↗
PubMed1988

Import of proteins into mitochondria.

A mounting body of evidence suggests that cytoplasmically synthesized proteins destined to be imported into the mitochondrial interior must at least partly unfold to penetrate across the mitochondrial membranes. During post-translational import, this unfolding process appears to be a major rate-limiting step. It can be blocked by ligands that stabilize the protein's native conformation and appears to be accompanied by the cleavage of ATP outside the mitochondrial inner membrane.

Animals↗
PubMed1988

Amphiphilicity is essential for mitochondrial presequence function.

We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non-amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this 'non-amphiphilic' presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non-amphiphilic. One of the active presequence peptides was non-helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.

Circular Dichroism↗
PubMed1988

Unfolding and refolding of a purified precursor protein during import into isolated mitochondria.

A purified mitochondrial precursor protein unfolds to a protease-sensitive conformation at the surface of isolated mitochondria before being imported into the organelles. This unfolding is stimulated by a potential across the mitochondrial inner membrane, but does not require ATP. In contrast, import of the surface-bound unfolded precursor requires ATP, but no potential; it is accompanied by a refolding inside the mitochondria.

Animals↗
PubMed1988

Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria.

In order to study the role of protein unfolding during post-translational protein import into mitochondria, we destabilized the structure of a mitochondrial precursor protein by site-directed mutagenesis. The precursor consisted of the first 16 residues of the yeast cytochrome oxidase subunit IV precursor fused to mouse dihydrofolate reductase. Labilization of the folded precursor structure was monitored by increased susceptibility to protease and diminished ability of methotrexate to block import of the precursor into isolated yeast mitochondria. On comparing the original precursor with two mutant forms that were destabilized to different degrees, increased labilization correlated with an increased rate and efficiency of import into mitochondria. This supports the view that the precursor must unfold in order to enter the mitochondria.

Animals↗
PubMed1988

Latent membrane perturbation activity of a mitochondrial precursor protein is exposed by unfolding.

We have purified milligram amounts of an importable mitochondrial precursor protein [the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (DHFR)]. This has made it possible, for the first time, to perform detailed studies on the conformation of a precursor protein and its interaction with lipid membranes. The precursor protein closely resembled authentic mouse DHFR with respect to secondary structure (measured by CD spectra) and stability towards urea (measured by tryptophan fluorescence and enzyme activity). With this precursor protein, the presequence thus does not significantly alter the folding of the attached 'passenger protein'. In contrast to the corresponding presequence peptide, the native precursor exhibited only weak ability to disrupt vesicles with a low mol% of negatively charged lipids, suggesting that the passenger protein masks the amphiphilic properties of the presequence. The membrane-perturbing properties of the precursor were greatly enhanced by increasing the vesicles' content of negatively charged lipid or by denaturing the precursor in 5 M urea. Interaction with vesicles rich in acidic phospholipid was accompanied by partial unfolding of the precursor, suggesting that such a conformational change may also be involved in the interaction of the precursor with the mitochondrial membranes.

Animals↗
PubMed1988

MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease.

We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.

Amino Acid Sequence↗