ABSTRACT
Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.
Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Acetyltransferases/genetics , Acetyltransferases/isolation & purification , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Cloning, Molecular , Conserved Sequence , Drosophila melanogaster/genetics , Histone Acetyltransferases , Humans , Macromolecular Substances , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Phenotype , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
Deoxyguanosine kinase (dGK) is a nuclear gene product that catalyzes the phosphorylation of purine deoxyribonucleosides and their analogues. The human enzyme is located predominantly in the mitochondria, as shown by biochemical fractionation studies and in situ localization of the overexpressed recombinant protein. Here we describe the cloning of mouse dGK cDNA and the identification of a novel amino-terminally truncated isoform that corresponds to about 14% of the total dGK mRNA population in mouse spleen. In situ fluorescence assays suggest that the new isoform cannot translocate into the mitochondria and thus may represent a cytoplasmic enzyme. Expression of mouse dGK mRNA was highly tissue-specific and differed from the tissue distribution observed in humans. Recombinant mouse dGK showed similar specific activity and substrate specificity as compared with the human enzyme. The broad specificity, restricted tissue distribution, and location of mouse dGK in multiple cellular compartments raise new considerations with respect to the role of the individual deoxynucleoside kinases in nucleotide metabolism.
Subject(s)
Phosphotransferases (Alcohol Group Acceptor)/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cloning, Molecular , Isoenzymes/genetics , Mice , Microscopy, Fluorescence , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/chemistry , RNA, Messenger/genetics , Sequence Deletion , Sequence Homology, Amino Acid , Substrate Specificity , TransfectionABSTRACT
Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxynucleoside salvage pathway in mammalian cells and plays a key role in the activation of several pharmacologically important nucleoside analogs. Using a highly specific polyclonal antibody raised against a C-terminal peptide of the human dCK, we analyzed its subcellular localization by Western blots of biochemically fractionated nuclear and cytoplasmic fractions as well as by in situ immunochemistry. Native dCK was found to be located mainly in the cytoplasm in several cell types, and the enzyme was more concentrated in the perinuclear and cellular membrane area. In contrast, when dCK was overexpressed in the cells, it was mainly located in the nucleus. The results demonstrate that native dCK is a cytoplasmic enzyme. However, it has the ability to enter the nucleus under certain conditions, suggesting the existence of a cytoplasmic retention mechanism that may have an important function in the regulation of the deoxynucleoside salvage pathway.
