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G Schatz

Publications and source records attributed to G Schatz.

At least 163 records · Page 9Linked to original sources
PubMed1986

The cleavable pre-sequence of an imported chloroplast protein directs attached polypeptides into yeast mitochondria.

The cleavable pre-sequences of imported chloroplast and mitochondrial proteins have several features in common. This structural similarity prompted us to test whether a chloroplast pre-sequence (;transit peptide') can also be decoded by the mitochondrial import machinery. In the green alga, Chlamydomonas reinhardtii, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (a chloroplast protein) is nuclear-encoded and synthesized in the cytosol with a transient pre-sequence of 45 residues. The 31 amino-terminal residues of this chloroplast pre-sequence were fused to mouse dihydrofolate reductase (a cytosolic protein) and to yeast cytochrome oxidase subunit IV (an imported mitochondrial protein) from which the authentic pre-sequence had been removed. The chloroplast pre-sequence transported both attached proteins into the yeast mitochondrial matrix or inner membrane, although it functioned less efficiently than an authentic mitochondrial pre-sequence. We conclude that mitochondrial and chloroplast pre-sequences perform their function by a similar mechanism.

Journal Article↗
PubMed1986

A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen.

The nuclear gene for manganese-containing superoxide dismutase (MnSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) of yeast mitochondria was mapped on chromosome VIII and inactivated by gene disruption. The resulting mutant lacked any protein cross-reacting with anti-MnSOD antibodies, and its mitochondria exhibited less than 1% of the cyanide-insensitive superoxide dismutase activity found in mitochondria of the wild-type parent strain. In the absence of oxygen, the mutant grew as rapidly as the wild-type parent. However, increasing concentrations of oxygen led to a progressive inhibition of growth. The properties of this mutant provide direct evidence that MnSOD contributes to the natural protection of cells against oxygen toxicity.

Arginine↗
PubMed1986

Artificial mitochondrial presequences.

Synthetic oligonucleotides were used to construct artificial mitochondrial presequences that contained, besides the initiator methionine, only arginine, serine, and leucine. The ratio of these three amino acids was adjusted to match that of basic, hydroxylated, and hydrophobic residues in natural mitochondrial presequences. When these sequences were fused to the N terminus of yeast cytochrome oxidase subunit IV lacking its own presequence, they directed the attached subunit IV to its correct intramitochondrial location in vivo. They also mediated import of subunit IV into isolated yeast mitochondria. In contrast, artificial sequences containing glutamine, arginine, and serine residues following the initiator methionine were inactive. Thus, the targeting function of mitochondrial presequences does not depend on specific amino acid sequences but may instead depend on the overall balance between basic, hydrophobic, and hydroxylated amino acids.

Amino Acid Sequence↗
PubMed1985

The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane.

We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre-sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane.

Biological Transport↗
PubMed1985

The cytoplasmically-made subunit IV is necessary for assembly of cytochrome c oxidase in yeast.

Yeast cytochrome c oxidase contains three large subunits made in mitochondria and at least six smaller subunits made in the cytoplasm. There is evidence that the catalytic centers (heme a and copper) are associated with the mitochondrially-made subunits, but the role of the cytoplasmically-made subunits has remained open. Using a gene interruption technique, we have now constructed a Saccharomyces cerevisiae mutant which lacks the largest of the cytoplasmically-made subunits (subunit IV). This mutant is devoid of cyanide-sensitive respiration, the absorption spectrum of cytochrome aa3 and cytochrome c oxidase activity. It still contains the other cytochrome c oxidase subunits but these are not assembled into a stable complex. Active cytochrome c oxidase was restored to the mutant by introducing a plasmid-borne wild-type subunit IV gene; no restoration was seen with a gene carrying an internal deletion corresponding to amino acid residues 28-66 of the mature subunit. Subunit IV is thus necessary for proper assembly of cytochrome c oxidase.

Cytoplasm↗
PubMed1985

The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix.

Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.

Amino Acid Sequence↗
PubMed1985

A yeast mutant temperature-sensitive for mitochondrial assembly is deficient in a mitochondrial protease activity that cleaves imported precursor polypeptides.

We have previously described two yeast mutants which, at elevated temperature, stop growing and accumulate precursors to several imported mitochondrial proteins. We now show that one of these mutants (mas 1) is deficient in a matrix-located protease activity which cleaves the pre-sequences from mitochondrial precursor proteins. Isolated mas 1 mitochondria catalyze oxidative phosphorylation, exhibit respiratory control and import mitochondrial precursor polypeptides, but are defective in removing transient pre-sequences from imported precursors. The phenotype of the mas 1 mutant suggests that the matrix-located processing protease is essential for growth and for mitochondrial assembly.

Adenosine Diphosphate↗
PubMed1984

The amino-terminal region of an imported mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix.

Subunit IV of yeast cytochrome c oxidase is encoded by a nuclear gene, synthesized in the cytosol as a precursor with a transient amino-terminal extension of 25 amino acids, and imported into the mitochondria. By gene fusion, we have attached the amino-terminal 53 amino acids of the subunit IV precursor to the amino terminus of the mouse cytosolic enzyme dihydrofolate reductase. When the resulting fusion protein was synthesized in a transcription-translation system and then incubated with energized yeast mitochondria, it was imported into the mitochondrial matrix space and processed to a shorter form by the chelator-sensitive matrix protease. No evidence was obtained that the fusion protein became stuck across one of the two mitochondrial membranes. Thus, a non-mitochondrial protein can be transported into the mitochondrial matrix if it is fitted with a mitochondrial targeting sequence.

Amino Acid Sequence↗
PubMed1984

A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus.

The major 70-kd protein of the yeast mitochondrial outer membrane is made on cytosolic ribosomes and imported into the outer membrane without proteolytic cleavage. We have attempted to identify the sequences which target the protein to the mitochondria and which permanently anchor it to the lipid bilayer of the outer membrane. By manipulating the cloned gene we have deleted 13 different regions throughout the polypeptide; in addition, we have fused amino-terminal regions of different length to beta-galactosidase. Each altered gene was introduced into yeast and the intracellular fate of the corresponding polypeptide product was determined by subcellular fractionation. All the information for targeting and anchoring the 70-kd protein (617 amino acids) was contained within the amino-terminal 41 amino acids. When this entire region was deleted, the protein was recovered with the cytosol fraction. However, several restricted deletions within this amino-terminal region appeared to affect targeting and anchoring differentially: most of the altered protein remained in the cytosol but a small fraction was misrouted into the mitochondrial matrix space. We suggest that targeting is mediated by a region which includes the 11 amino-terminal amino acids whereas the permanent membrane anchor is provided by a typical transmembrane sequence between residues 9 and 38.

Amino Acid Sequence↗
PubMed1984

The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix.

The cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.

Animals↗
PubMed1984

Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein.

The six small subunits (IV-VII, VIIa, VIII) of yeast cytochrome c oxidase are encoded by nuclear genes and imported into the mitochondria. We have isolated the gene for subunit IV from a yeast genomic clone bank and determined its complete nucleotide sequence. We have also isolated subunit IV from purified yeast cytochrome c oxidase and determined most of its amino acid sequence which confirms the positioning of approximately 90% of the amino acid residues. The sequence comparison shows that the coding sequence of the gene lacks introns and that subunit IV is made as a precursor with an amino-terminal extension of 25 residues, five of which are basic and none of them acidic. Precursor processing involves cleavage of a Leu-Gln bond.

Amino Acid Sequence↗
PubMed1984

A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria.

The beta-subunit of mitochondrial ATPase is coded by a nuclear gene, synthesized outside the mitochondria as a larger precursor and imported into mitochondria. The beta-subunit precursor was purified from yeast, both as a homogeneous, unlabeled polypeptide and in radiochemically pure form. Both precursor preparations were cleaved to the mature beta-subunit by partially purified processing protease from the mitochondrial matrix. However, import of the radiochemically pure precursor into isolated yeast mitochondria required a cytosolic fraction from yeast or reticulocytes. The cytosolic factor was non-dialyzable and trypsin-sensitive; its apparent mol. wt. was approximately 40 000 as judged by gel filtration. Import of some proteins into mitochondria thus requires proteins of the 'soluble' cytoplasm.

Biological Transport↗
PubMed1984

Isolation of the nuclear yeast genes for citrate synthase and fifteen other mitochondrial proteins by a new screening method.

To isolate nuclear genes specifying imported mitochondrial proteins, a yeast genomic clone bank was screened by an RNA hybridization-competition assay. This assay exploited the fact that mRNAs for imported mitochondrial proteins are enriched in polysomes which are bound to the mitochondrial surface in cycloheximide-inhibited yeast cells. Clones selectively hybridizing to these enriched mRNAs were further screened by hybrid-selected translation and immunoprecipitation with monospecific antisera against individual mitochondrial proteins. Thirty-six clones were isolated which contained complete or partial copies of 16 different genes for imported mitochondrial proteins. Several of these clones caused expression of the corresponding precursor polypeptide in Escherichia coli or over-expression of the corresponding mature protein in yeast. The gene for the matrix enzyme citrate synthase was sequenced; the derived amino acid sequence of the precursor polypeptide revealed an amino-terminal extension containing basic but no acidic residues.

Amino Acid Sequence↗
PubMed1984

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.

Amino Acid Sequence↗
PubMed1984

Two nuclear mutations that block mitochondrial protein import in yeast.

We isolated two yeast mutants that are temperature-sensitive for import of mitochondrial proteins. Each strain contains a single mutation that results in arrest of growth and accumulation of precursor to the beta subunit of the mitochondrial F1-ATPase after incubation at 37 degrees C. These lesions (mas1 and mas2) are nonallelic and recessive. Cells harboring either mutation stop growing only after 2-3 generations at 37 degrees C. Import of the F1 beta subunit at 37 degrees C is more than 250 times slower in mas1 and 15 times slower in mas2 than in wild-type cells. At 23 degrees C, import occurs with similar rates in mutant and wild-type cells. The two mutations also reduce the rate of import of other proteins; however, import of different precursors is affected to different degrees in the two strains. The temperature-sensitive step in import in both mas1 and mas2 occurs before arrival of precursors in the mitochondrial matrix.

Biological Transport↗
PubMed1983

Import of proteins into mitochondria. Partial purification of a matrix-located protease involved in cleavage of mitochondrial precursor polypeptides.

Most mitochondrial proteins are synthesized in the cytoplasm as larger precursor polypeptides which are imported into the organelle in an energy-dependent step. The proteolytic conversion of these precursors to their mature size involves a neutral matrix-located protease which has been purified 100-fold from yeast mitochondria. It cleaves the precursors to several imported proteins of the matrix, the mitochondrial inner membrane and the intermembrane space, but is inactive against all mature mitochondrial proteins or against all nonmitochondrial proteins tested so far. As shown in the subsequent report (Cerletti, N., Böhni, P. C., and Suda, K. (1983) J. Biol. Chem. 258, 4944-4949), processing of the cytochrome c oxidase subunit V precursor with the partially purified protease yielded the correct mature NH2 terminus. Precursors to cytochrome b2 and cytochrome c1 (which are imported into the intermembrane space and the outer face of the inner membrane, respectively) are cleaved to intermediate forms which can also be detected as transient forms in vivo. The protease activity has a pH optimum of 7.5 and is inhibited by 1,10-phenanthroline, EDTA, or nucleoside triphosphates, but not by serine-protease inhibitors or by small peptide inhibitors. Its activity can be restored after chelation by excess Co2+ or Zn2+. The enzyme is coded in the nucleus and is, thus, imported into mitochondria.

Amino Acid Sequence↗