Immunocytochemical evidence for the cytoplasmic localization and differential expression during the cell cycle of the M1 and M2 subunits of mammalian ribonucleotide reductase.
Mammalian ribonucleotide reductase consists of two non-identical subunits, proteins M1 and M2. We have produced and characterized rat polyclonal and monoclonal antibodies directed against protein M2 of mouse ribonucleotide reductase. Using these antibodies for immunocytochemical studies, an exclusively cytoplasmic localization of protein M2 was demonstrated both in cultured parent and hydroxyurea-resistant, M2-over-producing mouse TA3 cells, and in cells from various mouse tissues. These data, together with the previously demonstrated cytoplasmic localization of the M1 subunit, clearly show that ribonucleotide reductase is a cytoplasmic enzyme. Combining the anti-M2 antibodies with a monoclonal anti-M1 antibody allowed for double-labelling immunofluorescence studies of the two subunits in individual cells. Only approximately 50% of the cells in a logarithmically growing culture contained immunodetectable protein M2, while the M1-specific staining was present in all cells. The M2 staining correlates well with the proportion of cells in the S-phase of the cell cycle. In tissues, only actively dividing cells stained with either antibody and there were always fewer cells stained with the M2-antibodies than with the M1-antibody. Our data therefore present independent evidence for the earlier proposed model of a differential regulation during the cell cycle of the M1 and M2 subunits of ribonucleotide reductase.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002453:Cell Cycle; D003593:Cytoplasm / Q000032:analysis; D004790:Enzyme Induction; D005455:Fluorescent Antibody Technique; D051379:Mice; D012264:Ribonucleotide Reductases / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
D000911:Antibodies, Monoclonal; D012264:Ribonucleotide Reductases