A novel yeast gene coding for a putative protein kinase

A yeast gene termed YKR2 coding for a putative protein kinase was isolated using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein YKR2, containing 677 amino acids, shows about 40% sequence identity in the kinase region to a family of serine/threonine-specific protein kinases from various species.     

A yeast gene coding for a putative protein kinase homologous to cdc25 suppressing protein kinase

A yeast gene termed YKR coding for a putative protein kinase was isolated by using the cloned cDNA for rabbit protein kinase C as a hybridization probe. The encoded protein (YKR), composed of 380 amino acid residues, shows extensive sequence homology to serine/threonine-specific protein kinases from various species in the approx. 320 C-terminal amino acid residues, strongly suggesting that YKR is endowed with a protein kinase activity. The observed homologies to the cdc25 suppressing protein kinase from yeast, the catalytic subunit of mammalian cAMP-dependent protein kinase, and mammalian protein kinase C were 76, 48 and 37%, respectively. Gene replacement experiments showed that YKR itself is not essential for cell proliferation.     

ATP synthase complex from bovine heart mitochondria. Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein

Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0). In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea. The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two. However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase. The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential. The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide. Furthermore, OSCP does not prevent or block passive H+ leakage. Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems.     

MSW, a yeast gene coding for mitochondrial tryptophanyl-tRNA synthetase

E569 and E606 are noncomplementing pet mutants of Saccharomyces cerevisiae. Both strains are defective in mitochondrial protein synthesis and as a result exhibit a pleiotropic deficiency in respiratory components that are translated on mitochondrial ribosomes. The wild type gene MSW capable of complementing the protein synthesis defect has been cloned by transformation of one of the mutants with a genomic library of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein with a molecular weight of 42,414 which is 37 and 39% identical to the tryptophanyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus, respectively. A strain containing an insertion in the chromosomal copy of MSW was constructed by in situ gene replacement. This mutant fails to charge mitochondrial tryptophanyl-tRNA providing further evidence that MSW is the structural gene for mitochondrial tryptophanyl tRNA synthetase. The existence of another gene coding for the cytoplasmic tryptophanyl-tRNA synthetase is inferred from the observation that mutations in MSW are not lethal but only result in a respiratory deficiency.     

The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.

The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.     

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.     

Mapping the human gene coding for chromosomal protein HMG-17

Summary The functional gene coding for nonhistone chromosomal protein HMG-17, a nucleosomal binding protein that may confer unique properties to the chromatin structure of active genes, has been mapped to band 1p36.1. The multiple, nonfunctional, HMG-17 retropseudogenes are scattered over several chromosomes.     

KIN28, a yeast split gene coding for a putative protein kinase homologous to CDC28.

We have isolated, in yeast, a nuclear gene named KIN28 which presents significant sequence homology with the cell-division-cycle CDC28 gene, with members of the protein-tyrosine kinase family (src, erb, abl, epidermal growth factor, etc.) and those of the family of protein kinases phosphorylating serine and threonine. This strongly suggests that KIN28 is endowed with a protein kinase activity. In contrast with CDC28, KIN28 is interrupted by an intervening sequence. The KIN28 gene failed to complement cdc28 mutations and was shown to be essential for cell proliferation.     

Structure of the plasmodium knowlesi gene coding for the circumsporozoite protein

The gene that codes for the surface antigen of Plasmodium knowlesi sporozoites (CS protein) is unsplit and present in the genome in only one copy. The CS protein, as deduced from DNA sequence analysis of the structural gene, has an unusual structure with the central 40% of the polypeptide chain present as 12 tandemly repeated amino acid peptide units flanked by regions of highly charged amino acids. The protein has an amino-terminal hydrophobic amino acid signal sequence and a hydrophobic carboxy-terminal anchor sequence. The coding sequence of the gene has an AT content of 53%, compared with 70% AT in the 5′ and 3′ flanking sequences, and is contained entirely within an 11 kb Eco RI genomic DNA fragment. This genomic fragment expresses the CS protein in E. coli, indicating that the parasite promoter and ribosome binding site signals can be recognized in E. coli.     

The mouse Fgfrl1 gene coding for a novel FGF receptor-like protein

The mouse Fgfrl1 gene codes for a novel cell surface protein that is closely related to the family of the FGF receptors. It contains three extracellular Ig C2 loops and an acidic box, which share 29-33% sequence identity (48-50% similarity) with FGF receptors 1-4. The intracellular portion of the novel protein, however, lacks a tyrosine kinase domain required for signal transduction by transphosphorylation. The gene for Fgfrl1 comprises six exons and is located on mouse chromosome 5 in close proximity to the Idua gene for L-iduronidase.     

Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

Pig heart mitochondrial membranes depleted of F1 and OSCP by various treatments were analyzed for their content in alpha and beta subunits of F1 and in OSCP using monoclonal antibodies. Membrane treatments and conditions of rebinding of F1 and OSCP were optimized to reconstitute efficient NADH- and ATP-dependent proton fluxes, ATP synthesis and oligomycin-sensitive ATPase activity. F1 and OSCP can be rebound independently to depleted membranes but to avoid unspecific binding of F1 to depleted membranes (ASUA) which is not efficient for ATP synthesis, F1 must be rebound before the addition of OSCP. The rebinding of OSCP to depleted membranes reconstituted with F1 inhibits the ATPase activity of rebound F1, while it restores the ATP-driven proton flux measured by the quenching of ACMA fluorescence. The rebinding of OSCP also renders the ATPase activity of bound F1 sensitive to uncouplers. The rebinding of OSCP alone or F1 alone, does not modify the NADH-dependent proton flux, while the rebinding of both F1 and OSCP controls this flux, inducing an inhibition of the rate of NADH oxidation. Similarly, oligomycin, which seals the F0 channel even in the absence of F1 and OSCP, inhibits the rate of NADH oxidation. OSCP is required to adjust the fitting of F1 to F0 for a correct channelling of protons efficient for ATP synthesis. All reconstituted energy-transfer reactions reach their optimal value for the same amount of OSCP. This amount is consistent with a stoichiometry of two OSCP per F1 in the F0-F1 complex.     

Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulphur protein

We have previously reported the isolation of the gene coding for a 25-kDa polypeptide present in a purified yeast QH2:cytochrome c oxidoreductase preparation, which was thus identified as the gene for the Rieske iron-sulphur protein [Van Loon et al. (1983) Gene 26, 261-272]. Subsequent DNA sequence analysis reported here reveals, however, that the encoded protein is in fact manganese superoxide dismutase, a mitochondrial matrix protein. Comparison with the known amino acid sequence of the mature protein indicates that it is synthesized with an N-terminal extension of 27 amino acids. In common with the N-terminal extensions of other imported mitochondrial proteins, the presequence has several basic residues but lacks negatively charged residues. The function of these positive charges and other possible topogenic sequences are discussed. Sequences 5' of the gene contain two elements that may be homologous to the suggested regulatory sites, UAS 1 and UAS 2 in the yeast CYC1 gene [Guarente et al. (1984) Cell 36, 503-511]. The predicted secondary structures in manganese superoxide dismutase appear to be very similar to those reported for iron superoxide dismutase, suggesting similar three-dimensional structures. Making use of the known three-dimensional structure of the Fe enzyme, the Mn ligands are predicted.     

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.     

Purification and identification of an estrogen binding protein from rat brain: oligomycin sensitivity-conferring protein (OSCP), a subunit of mitochondrial F0F1-ATP synthase/ATPase

Early studies have suggested the presence in the central nervous system of possible estrogen binding sites/proteins other than classical nuclear estrogen receptors (nER). We report here the isolation and identification of a 23 kDa membrane protein from digitonin-solubilized rat brain mitochondrial fractions that binds 17beta-estradiol conjugated to bovine serum albumin at C-6 position (17beta-E-6-BSA), a ligand that also specifically binds nER. This protein was partially purified using affinity columns coupled with 17beta-E-6-BSA and was recognized by the iodinated 17beta-E-6-BSA (17beta-E-6-[125I]BSA) in a ligand blotting assay. The binding of 17beta-E-6-BSA to this protein was specific for the 17beta-estradiol portion of the conjugate, not BSA. Using N-terminal sequencing and immunoblotting, this 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP). This protein is a subunit of the FOF1 (F-type) mitochondrial ATP synthase/ATPase required for the coupling of a proton gradient across the F0 sector of the enzyme in the mitochondrial membrane to ATP synthesis in the F1 sector of the enzyme. Studies using recombinant bovine OSCP (rbOSCP) in ligand blotting revealed that rbOSCP bound 17beta-E-6-[125I]BSA with the same specificity as the purified 23 kDa protein. Further, in a ligand binding assay, 17beta-E-6-[125I]BSA also bound rbOSCP and it was displaced by both 17beta-E-6-BSA and 17alpha-E-6-BSA as well as partially by 17beta-estradiol and diethylstilbestrol (DES), but not by BSA. This finding opens up the possibility that estradiol, and probably other compounds with similar structures, in addition to their classical genomic mechanism, may interact with ATP synthase/ATPase by binding to OSCP, and thereby modulating cellular energy metabolism. Current experiments are addressing such an issue.