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G Schatz

Publications and source records attributed to G Schatz.

At least 73 records · Page 4Linked to original sources
PubMed1994

Incomplete arrest in the outer membrane sorts NADH-cytochrome b5 reductase to two different submitochondrial compartments.

The S. cerevisiae gene MCR1 encodes two mitochondrial isoforms of NADH-cytochrome b5 reductase. The primary translation product has an amino-terminal matrix-targeting signal, followed by a stretch of 21 uncharged amino acids. This precursor protein is inserted into the outer membrane, but only about one-third of the molecules become firmly anchored to the outer face of that membrane. The remaining molecules pass through the outer membrane into the inner membrane, are cleaved by inner membrane protease 1, and are released into the intermembrane space. Incomplete translocation arrest in the outer membrane is a novel mechanism by which the product of a single gene is sorted into different compartments of the same organelle.

Amino Acid Sequence↗
PubMed1994

Fusion proteins containing the cytochrome b2 presequence are sorted to the mitochondrial intermembrane space independently of hsp60.

hsp60 is a chaperonin located in the mitochondrial matrix. It has been suggested that hsp60 participates in two processes: protein folding in the matrix, and the sorting of imported proteins to the intermembrane space. We analyzed hsp60 function by allowing isolated mitochondria to import two model precursor proteins and then measuring the binding of these proteins to the chaperonin. Of the methods that we tested for monitoring the association of imported proteins with hsp60, only co-immunoprecipitation with specific anti-hsp60 antibodies proved to be reliable. A chimeric matrix-targeted precursor, consisting of a mitochondrial presequence fused to a chloroplast-encoded protein, bound stably to hsp60 after import. In contrast, there was no detectable binding to hsp60 with a fusion protein that was targeted to the intermembrane space by the bipartite cytochrome b2 presequence. Analysis of a translocation intermediate demonstrated that the cytochrome b2 presequence arrests import through the inner membrane, with the result that the attached passenger protein is never exposed to hsp60.

Biological Transport↗
PubMed1994

Yeast mitochondria lacking the two import receptors Mas20p and Mas70p can efficiently and specifically import precursor proteins.

Mas20p and Mas70p are integral proteins of the yeast mitochondrial outer membrane that appear to function as receptors for precursor proteins imported from the cytosol. Loss of either receptor alone does not block import or kill the cells, but deletion of Mas20p causes loss of respiration (Ramage, L., Junne, T., Hahne, K., Lithgow, T., and Schatz, G. (1993) EMBO J. 12, 4115-4123). Here we show that this respiratory deficiency is only temporary; given time to adapt, virtually all cells lacking MAS20p regain respiration without regaining MAS20p. The respiratory defect can also be suppressed (at a frequency of about 10(-6)) by a dominant mutation of a single nuclear gene. The suppressed cells, unlike the unsuppressed ones, tolerate disruption of the MAS70 gene. The resulting double disruptants lack both Mas20p and Mas70p, yet are viable and able to respire. Protein import into mitochondria isolated from these cells is efficient, specific, and highly sensitive to protease treatment. We propose that at least one additional mitochondrial surface protein can function as a protein import receptor and that the activity of this component is up-regulated by a stress response or by an extragenic suppressor.

Biological Transport↗
PubMed1994

A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability.

Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C-terminal hexa-histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel-derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli.

Adenosine Triphosphate↗
PubMed1994

Requirement for the yeast gene LON in intramitochondrial proteolysis and maintenance of respiration.

The role of protein degradation in mitochondrial homeostasis was explored by cloning of a gene from Saccharomyces cerevisiae that encodes a protein resembling the adenosine triphosphate (ATP)-dependent bacterial protease Lon. The predicted yeast protein has a typical mitochondrial matrix-targeting sequence at its amino terminus. Yeast cells lacking a functional LON gene contained a nonfunctional mitochondrial genome, were respiratory-deficient, and lacked an ATP-dependent proteolytic activity present in the mitochondria of Lon+ cells. Lon- cells were also impaired in their ability to catalyze the energy-dependent degradation of several mitochondrial matrix proteins and they accumulated electron-dense inclusions in their mitochondrial matrix.

ATP-Dependent Proteases↗
PubMed1994

Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.

Adenosine Triphosphate↗
PubMed1993

Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E. coli groES.

The mitochondrial chaperonin system consists of chaperonin 60 (also termed hsp60), which is homologous to E. coli groEL, and chaperonin 10, which is homologous to E. coli groES. In yeast, chaperonin 60 function has been shown to be essential for viability. We report here that the same is true for chaperonin 10. We have cloned, sequenced and disrupted the nuclear chaperonin 10 gene CPN10 from Saccharomyces cerevisiae. This gene encodes a protein of 11,372 Da that is imported into the mitochondrial matrix without detectable cleavage. Haploid cells lacking a functional copy of CPN10 fail to grow at temperatures between 23 and 37 degrees C.

Amino Acid Sequence↗
PubMed1993

Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria.

Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.

Adenosine Triphosphatases↗
PubMed1993

Precursor binding to yeast mitochondria. A general role for the outer membrane protein Mas70p.

Binding of precursors to import receptors on the mitochondrial surface is one of the earliest steps of protein import into mitochondria. In yeast, one of these receptors is a 70-kDa outer membrane protein termed Mas70p. Pulse-chase studies with intact yeast cells had indicated that Mas70p accelerates the import of all mitochondrial precursors tested. In contrast, import experiments with isolated mitochondria suggested that Mas70p accelerated import of only a subset of precursors (Hines, V., Brandt, A., Griffiths, G., Horstmann, H., Brütsch, H., and Schatz, G. (1990) EMBO J. 9, 3191-3200). To resolve this discrepancy, we have now studied the interaction of Mas70p-deficient and wild-type yeast mitochondria with a precursor (pre-alcohol dehydrogenase III) whose import into isolated mitochondria is not accelerated by Mas70p under the usual assay conditions. Mas70p enhanced binding of pre-alcohol dehydrogenase III to the surface of mitochondria in which the electrochemical potential across the inner membrane had been dissipated by an uncoupler; the bound precursor could be efficiently chased into the mitochondria if the potential was restored. The precursor to cytochrome c1 was also bound to mitochondria in a Mas70p-dependent manner. Mas70p also enhanced the direct import of pre-alcohol dehydrogenase III into isolated mitochondria, provided the precursor was first denatured with urea. Under these conditions, the import rate in vitro was more similar to that in intact cells. Mas70p had no effect on the binding or the import of artificial precursors containing mouse dihydrofolate as the "mature" domain. We conclude that Mas70p is an import receptor for most, if not all authentic mitochondrial precursor proteins, but that its function is not always rate-limiting in import experiments with isolated mitochondria.

Alcohol Dehydrogenase↗
PubMed1993

The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions.

Cytochrome b2, a protein of the yeast mitochondrial intermembrane space, is synthesized with an 80 residue bipartite presequence. The amino-terminal portion resembles a matrix-targeting signal. The carboxy-terminal portion acts as a 'sorting signal' for the intermembrane space and contains a hydrophobic stretch. In order to define this sorting signal, we fused the first 167 residues of the cytochrome b2 precursor to a passenger protein, expressed the fusion protein in yeast and selected for mutations that caused mislocalization of the passenger protein to the matrix. Most mutations mapped within the first 81 amino-terminal residues of the cytochrome b2 moiety. They were located in three regions, all downstream of the matrix-targeting domain: a cluster of three basic residues upstream of the hydrophobic stretch, the hydrophobic stretch itself and the first residue of mature cytochrome b2. The level of missorting caused by mutations within the hydrophobic stretch did not correlate with their effects on hydrophobicity, but appeared to be related to changes in the conformation of this stretch. We conclude that the intermembrane space sorting signal of cytochrome b2 is decoded by protein-protein interactions rather than by simple partitioning into a lipid bilayer.

Amino Acid Sequence↗
PubMed1993

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1993

Functional cooperation of mitochondrial protein import receptors in yeast.

We have identified a 20 kDa yeast mitochondrial outer membrane protein (termed MAS20) which appears to function as a protein import receptor. We cloned, sequenced and physically mapped the MAS20 gene and found that the protein is homologous to the MOM19 import receptor from Neurospora crassa. MAS20 and MOM19 contain the sequence motif F-X-K-A-L-X-V/L, which is repeated several times with minor variations in the MAS70/MOM72 receptors. To determine how MAS20 functions together with the previously identified yeast receptor MAS70, we constructed yeast mutants lacking either one or both of the receptors. Deletion of either receptor alone had little or no effect on fermentative growth and only partially inhibited mitochondrial protein import in vivo. Deletion of both receptors was lethal. Deleting only MAS70 did not affect respiration; deleting only MAS20 caused loss of respiration, but respiration could be restored by overexpressing MAS70. Import of the F1-ATPase beta-subunit into isolated mitochondria was only partly inhibited by IgGs against either MAS20 or MAS70, but both IgGs inhibited import completely. We conclude that the two receptors have overlapping specificities for mitochondrial precursor proteins and that neither receptor is by itself essential.

Amino Acid Sequence↗
PubMed1993

Import of cytochrome b2 to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP.

Cytochrome b2 is synthesized as a precursor in the cytoplasm and imported to the intermembrane space of yeast mitochondria. We show here that the precursor contains a tightly folded heme-binding domain and that translocation of this domain across the outer membrane requires ATP. Surprisingly, it is ATP in the mitochondrial matrix rather than external ATP that drives import of the heme-binding domain. When the folded structure of the heme-binding domain is disrupted by mutation or by urea denaturation, import and correct processing take place in ATP-depleted mitochondria. These results indicate that (1) cytochrome b2 reaches the intermembrane space without completely crossing the inner membrane, and (2) some precursors fold outside the mitochondria but remain translocation-competent, and import of these precursors in vitro does not require ATP-dependent cytosolic chaperone proteins.

Adenosine Triphosphate↗