IDENTIFICATION OF FUNCTIONAL DOMAINS IN THE SEP1 PROTEIN (=KEM1, XRN1), WHICH IS REQUIRED FOR TRANSITION THROUGH MEIOTIC PROPHASE IN SACCHAROMYCES-CEREVISIAE

The Sep1 (also known as Kem1, Xrn1, Rar5, DST2/Stp beta) protein of Sa ccharomyces cerevisiae is an Mr 175,000 multifunctional exonuclease wi th suspected roles in RNA turnover and in the microtubular cytoskeleto n as well as in DNA recombination and DNA replication. The most striki ng phenotype of SEP1 null mutations is quantitative arrest during meio tic prophase at the pachytene stage. We have constructed a set of N- a nd C-terminal as well as internal deletions of the large SEP1 gene. An alysis of these deletion mutations on plasmids in a host carrying a nu ll allele (sep1 Delta) revealed that at least 270 amino acids from the C-terminus of the wild-type protein were dispensable for complementin g the slow growth and benomyl hypersensitivity of a null mutant. In co ntrast, any deletion at the N-terminus abrogated complementing activit y for these phenotypes. The sequences essential for function correspon d remarkably well with the regions of Sep1 that are homologous to its Schizosaccharomyces pombe counterpart Exo2. In addition, these experim ents showed that, despite the high intracellular levels of Sep1, over- expression of this protein above these levels is detrimental to the ce ll. We discuss the potential cellular roles of the Sep1 protein as a m icrotubule-nucleic acid interface protein linking its suspected functi on in the microtubular cytoskeleton with its role as a nucleic acid bi nding protein.