Stimulation of in vitro mitochondrial protein synthesis by yeast cytoplasmic extracts is caused by guanyl nucleotides

Micromolar concentrations of GDP or GTP stimulate protein synthesis by isolated yeast mitochondria 3- to 10-fold even if alpha-ketoglutarate and an ATP-regenerating system are present. No stimulation is observed with GMP, UTP, CTP, TTP, and the nonhydrolyzable GTP analogues guanyl(beta, gamma-methylene) diphosphate and guanyl imidodiphosphate. This stimulatory effect of exogenously added guanyl nucleotides may answer the long standing question why protein synthesis by isolated mitochondria is so slow. It can also explain previous reports by two other laboratories that a high speed supernatant from yeast cells stimulates protein synthesis by isolated mitochondria. The supernatant contains nondialyzable GMP which is converted to GDP under the conditions used for assaying mitochondrial protein synthesis. The stimulatory effect of high speed supernatants is abolished by 5'-nucleotidase (which degrades GMP) or by trypsin (which destroys supernatant protein(s) necessary for converting GMP to GDP). No evidence was obtained that the stimulatory effect of high speed supernatants was caused by precursors to cytoplasmically made cytochrome c oxidase subunits.     

Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.     

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.     

The MAS-encoded processing protease of yeast mitochondria. Interaction of the purified enzyme with signal peptides and a purified precursor protein

The matrix of yeast mitochondria contains a chelator-sensitive protease that removes matrix-targeting signals from most precursor proteins transported into this compartment. The enzyme consists of two nonidentical subunits that are encoded by the nuclear genes MAS1 and MAS2. With the aid of these cloned genes, we have now overexpressed the active holoenzyme in yeast, purified it in milligram amounts, and studied its biochemical and physical properties. Atomic absorption analysis shows that the purified enzyme lacks significant amounts of zinc, manganese, or cobalt; if none of these metal ions is added during the assay, the enzyme is catalytically inactive but can still cleave substoichiometric amounts of substrate. The amino-terminal sequences of the two mature subunits were determined; comparison with the deduced amino acid sequences of the corresponding precursors revealed that the MAS1 and MAS2 subunits are synthesized with prepeptides composed of 19 and 13 residues, respectively, which have similar sequences. The enzyme is inhibited competitively by chemically synthesized matrix-targeting peptides; the degree of inhibition correlates with the peptides' targeting efficacy. Matrix-targeting peptides containing the cleavage site of the corresponding authentic precursor protein are cleaved correctly by the purified enzyme. A purified artificial precursor protein bound to the holoenzyme can be photocross-linked to the MAS2 subunit.     

Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria

Import of in vitro-synthesized cytochrome b2 (a soluble intermembrane space enzyme) was studied wih isolated yeast mitochondria. Import requires an electrochemical gradient across the inner membrane and is accompanied by cleavage of the precursor to the corresponding mature form. This conversion proceeds via an intermediate form of cytochrome b2, which can be detected as a transient species when mitochondria are incubated with the cytochrome b2 precursor for short times or at low temperatures. Conversion of the precursor to the intermediate form is energy-dependent and catalyzed by an o-phenanthroline-sensitive protease located in the soluble matrix. The intermediate is subsequently converted to mature cytochrome b2 in a reaction which is o-phenanthroline-insensitive and requires neither an energized inner membrane nor a soluble component of the intermembrane space. Whereas mature cytochrome b2 is soluble, the intermediate formed by isolated mitochondria is membrane-bound and exposed to the intermembrane space. The same intermediate is detected as a transient species during cytochrome b2 maturation in intact yeast cells (Reid, G. A., Yonetani, T., and Schatz, G (1982) J. Biol. Chem. 257, 13068-13074). The in vitro studies reported here suggest that a part of the cytochrome b2 precursor polypeptide chain is transported to the matrix where it is cleaved to a membrane-bound intermediate form by the same protease that processes polypeptides destined for the matrix space or for the inner membrane. In a second reaction, the cytochrome b2 intermediate is converted to mature cytochrome b2 which is released into the intermembrane space. The binding of heme is not necessary for converting the intermediate to the mature polypeptide.     

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.     

The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting

Gene fusion experiments were used to identify signals that direct imported precursor proteins to specific intramitochondrial locations in yeast. The amino terminus of alcohol dehydrogenase III (ADHIII, a mitochondrial matrix enzyme) transported attached mouse dihydrofolate reductase (DHFR, a cytosolic enzyme) into the mitochondrial matrix. The presequence of cytochrome c1 (a mitochondrial inner membrane protein protruding into the intermembrane space) transported attached DHFR into the intermembrane space. The first half of the cytochrome c1 presequence, which resembles the ADHIII presequence, is a matrix-targeting sequence: it transported attached DHFR into the matrix. The second half of the cytochrome c1 presequence contains a stretch of 19 uncharged amino acids and may thus be a stop-transfer sequence. We conclude that intramitochondrial sorting involves matrix-targeting and stop-transfer sequences within the cleavable presequence.     

Effects of interaction between estradiol-17 beta and progesterone on the proliferation of cloned breast tumor cells (MCF-7 and T47D)

We have previously reported that the proliferation of cloned MCF-7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal-dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 X 10(-11) M estradiol-17 beta to the culture media. These observations suggest that the proliferation of T47D and MCF-7 cells is regulated by a blood-borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF-7 cells were grown in DME containing 2-40% CDFHS. Progesterone, at 3 X 10(-7) M to 3 X 10(-12) M, had no effect on the yield of MCF-7 or T47D cells that were cultured in the presence or absence of estradiol-17 beta.