Hsl7p, the yeast homologue of human JBP1, is a protein methyltransferase

The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase. In this report, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [(3)H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not. We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1. These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology.     

Cytochrome c1 of bakers' yeast. I. Isolation and properties.

Cytochrome c1 has been purified from mitochondria of the yeast Saccharomyces cerevisiae. The procedure involves solubilization withcholate, ammonium sulfate fractionation, disruption of the dytochrome b-c1 complex with mercaptoethanol and detergents, and chromatography on DEAE-cellulose. The final product is psectrally pure, contains up to 62 nmol of covalently bound heme per mg of protein and does not react with oxygen or carbon monoxide. Sodium dodecyl sulfate disaggregates the purified cytochrome into a single 31,000 dalton subunit carrying the covalently attached heme group. Many cytochrome c1 preparations contain in addition an 18,500 dalton polypeptide which is devoid of covalently bound heme. Since this polypeptide can be removed from the heme-carrying polypeptide by relatively mild procedures, it is probably not an essential subunit of cytochrome c1. Cytochrome c1 is extremely sensitive to proteolysis. If it si purified in the absence of protease inhibitors, a family of heme polypeptides with molecular weights of 29,000, 27,000, and 25,000 daltons is obtained. In the presence of the protease inhibitor phenylmethylsulfonylfluoride the purification yields predominantly a 31,000 dalton heme protein with only little contamination by a 29,000 dalton degradation product. In order to show that only the 31,000 dalton heme-polypeptide is the native species, yeast cells were labeled with the heme-precursor delta-amino[3H]levulinic acid, converted to protoplasts and directly lysed with dodecyl sulfate in the presence of protease inhibitors. Subsequent electrophoresis of the lysate in the presence of dodecyl sulfate reveals the covalently bound heme of cytochrome c1 as a single symmetrical peak at 31,000 daltons.     

Cytochrome c1 complexes.

Cytochrome c1 forms an active complex with cytochrome c as previously reported (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36). It also forms a complex with cytochrome oxidase with heme ratio of 1:1. This cytochrome c1.oxidase complex has been purified by ammonium sulfate fractionation and is stable in media of high ionic strength (greater than 0.1 M) but dissociates as the pH deviates from neutral. The purified cytochrome c1 aggregates to an oligomer, presumably a pentamer. No agent has been found to depolymerize isolated c1 without denaturation. However, in the cytochrome c1.oxidase complex, these two cytochromes apparently were depolymerized to form smaller aggregates, if not monomeric units, as judged by sedimentation behavior. Cytochrome c1 also forms a ternary complex with cytochrome c and oxidase in the heme ratio of 1:1:1. This complex can be prepared by any of the following four methods: (i) c1 + c + oxidase: (ii) c1.c complex + oxidase; (iii) c1 + c.oxidase complex: or (iv) c + c1.oxidase complex. The mode of formation of these complexes is all from pure protein-protein interactions. Cytochrome c1 is also incorporated into phospholipid vesicles and these vesicles show about 200 molecules of phospholipid/cytochrome c1 in terms of heme. The spectrophotometric, circular dichroic, sedimentation behavior and enzymic properties of these complexes have been investigated.     

Cytochrome c1 from Paracoccus denitrificans

Cytochrome c1 was purified from the bacterium Paracoccus denitrificans. It is an acidic, hydrophobic polypeptide with an apparent molecular weight of around 65000 and a single, covalently attached heme; it cross-reacts immunologically with cytochrome c1 from yeast mitochondria. The amino acid sequence of the tryptic heme peptide of the bacterial cytochrome c1 shows extensive homology to the corresponding region of beef heart cytochrome c1 [Wakabayashi, S. et al. (1982) J. Biol. Chem. 257, 9335-9344]. Positive evidence for a stable association of the Paracoccus cytochrome c1 with other polypeptides and b-type heme components ('bc1-complex') has not yet been obtained.     

Kinetics of Electron Transfer within Cytochrome bc 1 and Between Cytochrome bc 1 and Cytochrome c

In this minireview an overview is presented of the kinetics of electron transfer within the cytochrome bc (1) complex, as well as from cytochrome bc (1) to cytochrome c. The cytochrome bc (1) complex (ubiquinone:cytochrome c oxidoreductase) is an integral membrane protein found in the mitochondrial respiratory chain as well as the electron transfer chains of many respiratory and photosynthetic bacteria. Experiments on both mitochondrial and bacterial cyatochrome bc (1) have provided detailed kinetic information supporting a Q-cycle mechanism for electron transfer within the complex. On the basis of X-ray crystallographic studies of cytochrome bc (1), it has been proposed that the Rieske iron-sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c (1). A new method was developed to study electron transfer within cytochrome bc (1) using a binuclear ruthenium complex to rapidly photooxidize cytochrome c (1). The rate constant for electron transfer from the iron-sulfur center to cytochrome c (1) was found to be 80,000 s(-1), and is controlled by the dynamics of conformational changes in the iron-sulfur protein. Moreover, a linkage between the conformation of the ubiquinol binding site and the conformational dynamics of the iron-sulfur protein has been discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method has also been developed to measure electron transfer from cytochrome c (1) to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc (1) and cytochrome c.     

In vivo analysis of nucleolar proteins modified by the yeast arginine methyltransferase Hmt1/Rmt1p

In this report, we have investigated the impact of arginine methylation on the Gar1, Nop1, and Nsr1 nucleolar proteins in Saccharomyces cerevisiae. Although previous reports have established that protein arginine methylation is important for nucleocytoplasmic shuttling, they have focused on the examination of heterogeneous nuclear ribonucleoproteins (hnRNPs). We have extended this analysis to several nucleolar proteins that represent a distinct functional class of arginine-methylated proteins. We first developed an in vivo assay to identify proteins methylated by the Hmt1 arginine methyltransferase. This assay is based on the fact that the Hmt1 enzyme utilizes S-Adenosyl-L-methionine as the methyl donor for protein arginine methylation. Following SDS polyacrylamide electrophoresis, 11 distinct proteins were identified as substrates for the Hmt1 methyltransferase. Hmt1p overexpression did not increase the methylation level on these proteins, suggesting they are fully methylated under the conditions examined. Three of the radiolabeled proteins were confirmed to be Gar1p, Nop1p, and Nsr1p. To monitor the cellular localization of these proteins, functional GFP fusion proteins were generated and found to be localized to the nucleolus. This localization was independent of arginine methylation. Furthermore, all three proteins examined did not export to the cytoplasm. In contrast, arginine methylation is required for the export of the nuclear RNA-binding proteins Npl3p, Hrp1p, and Nab2p. The observation that three nucleolar proteins are modified by Hmt1p but are not exported from the nucleolus implies an alternate role for arginine methylation.     

The structure and oligomerization of the yeast arginine methyltransferase, Hmt1

Protein methylation at arginines is ubiquitous in eukaryotes and affects signal transduction, gene expression and protein sorting. Hmt1/Rmt1, the major arginine methyltransferase in yeast, catalyzes methylation of arginine residues in several mRNA-binding proteins and facilitates their export from the nucleus. We now report the crystal structure of Hmt1 at 2.9 A resolution. Hmt1 forms a hexamer with approximate 32 symmetry. The surface of the oligomer is dominated by large acidic cavities at the dimer interfaces. Mutation of dimer contact sites eliminates activity of Hmt1 both in vivo and in vitro. Mutating residues in the acidic cavity significantly reduces binding and methylation of the substrate Npl3.     

Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase

Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The elongated structure of the conserved core of yeast Dot1p contains an N-terminal helical domain and a seven-stranded catalytic domain that harbors the binding site for the methyl-donor and an active site pocket sided with conserved hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended conformation distinct from the folded conformation observed in structures of SET domain histone lysine methyltransferases. A catalytic asparagine (Asn479), located at the bottom of the active site pocket, suggests a mechanism similar to that employed for amino methylation in DNA and protein glutamine methylation. The acidic, concave cleft between the two domains contains two basic residue binding pockets that could accommodate the outwardly protruding basic side chains around Lys79 of histone H3 on the disk-like nucleosome surface. Biochemical studies suggest that recombinant Dot1 proteins are active on recombinant nucleosomes, free of any modifications.     

线粒体、细胞色素c与细胞凋亡

线粒体是细胞的一个独特而重要的细胞器,它为细胞各种生命活动提供能量。许多研究表明,线粒体的作用远比人们了解的复杂和多样。近年来研究发现,线粒体与细胞凋亡密切相关,表现在如下一些方面。１．细胞凋亡早期线粒体跨膜电位（ΔΨｍ）下降自１９９３年以来,Ｋｒｏ...     

线粒体,细胞包素c与细胞凋亡

线粒体是细胞的一个独特而重要的细胞器,它为细胞各种生命活动提供能量.许多研究表明,线粒体的作用远比人们了解的复杂和多样.近年来研究发现,线粒体与细胞凋亡密切相关,表现在如下一些方面.     

Analysis of the yeast arginine methyltransferase Hmt1p/Rmt1p and its in vivo function. Cofactor binding and substrate interactions

Many eukaryotic RNA-binding proteins are modified by methylation of arginine residues. The yeast Saccharomyces cerevisiae contains one major arginine methyltransferase, Hmt1p/Rmt1p, which is not essential for normal cell growth. However, cells missing HMT1 and also bearing mutations in the mRNA-binding proteins Npl3p or Cbp80p can no longer survive, providing genetic backgrounds in which to study Hmt1p function. We now demonstrate that the catalytically active form of Hmt1p is required for its activity in vivo. Amino acid changes in the putative Hmt1p S-adenosyl-L-methionine-binding site were generated and shown to be unable to catalyze methylation of Npl3p in vitro and in vivo or to restore growth to strains that require HMT1. In addition these mutations affect nucleocytoplasmic transport of Npl3p. A cold-sensitive mutant of Hmt1p was generated and showed reduced methylation of Npl3p, but not of other substrates, at 14 degrees C. These results define new aspects of Hmt1 and reveal the importance of its activity in vivo.     

Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth⁺) in the absence of catalase activity. These Mth⁺ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H₂O₂ does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H₂O₂-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized. Received: 9 December 1996 / Received revision: 5 May 1997 / Accepted: 25 May 1997     

hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Cytochrome c methylation

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.     

Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p

In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.     

Cytochrome c oxidase, ligands and electrons

We present hereby an overview of the reactions of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, with ligands (primarily oxygen) and electrons, pointing out where necessary unresolved facts or questionable interpretations.