THE MULTIFUNCTIONAL PROTEIN OBF1 IS PHOSPHORYLATED AT SERINE AND THREONINE RESIDUES IN SACCHAROMYCES-CEREVISIAE

We have purified a DNA replication enhancer-binding protein, OBF1, from yeast cells grown in a medium containing P-32-labeled orthophosphate. The purified P-32-labeled protein comigrated on polyacrylamide gels with OBF1 bands identified by immunoblotting with anti-OBF1 antibodies. Furthermore, trypsin treatment of the P-32-labeled OBF1 revealed several phosphorylated peptides, suggesting that OBF1 is multiply phosphorylated in vivo. Incubation of phosphorylated peptides with calf intestinal phosphatase liberated the radiolabel as free phosphate, indicating a phosphoester linkage. Acid hydrolysis of the tryptic peptides revealed P-32 label comigrating with phosphoserine; some of it, however, was also identified as phosphothreonine. Using anti-OBF1 antibodies, we cloned the OBF1 gene from a lambda-gt11 yeast expression library. The DNA sequence of the isolated gene and its over-expression in yeast indicated that OBF1 is identical to ABF-I and BAF1 proteins, believed to have a role in transcriptional repression and activation. Therefore, we suggest that OBF1 is a multifunctional protein, acting in transcription and replication, and that these activities are regulated by phosphorylation.