THE 110-KD SPINDLE POLE BODY COMPONENT OF SACCHAROMYCES-CEREVISIAE ISA PHOSPHOPROTEIN THAT IS MODIFIED IN A CELL CYCLE-DEPENDENT MANNER

Spc110p (Nuf1p) is an essential component of the yeast microtubule org anizing center, or spindle pole body (SPB). Asynchronous wild-type cul tures contain two electrophoretically distinct isoforms of Spc110p as detected by Western blot analysis, suggesting that Spc110p is modified in vivo. Both isoforms incorporate P-32(i) in vivo, suggesting that S pc110p is post-translationally modified by phosphorylation. The slower -migrating 120-kD Spc110p isoform is converted to the faster-migrating 112-kD isoform after incubation with protein phosphatase PP2A, and sp ecific PP2A inhibitors block this conversion. Thus, additional phospho rylation of Spc110p at serine and/or threonine residues gives rise to the slower-migrating 120-kD isoform. The 120-kD isoform predominates i n cells arrested in mitosis by the addition of nocodazole. However, th e 120-kD isoform is not detectable in cells grown to stationary phase (G0) or in cells arrested in G1 by the addition of alpha-factor. Tempe rature-sensitive cell division cycle (cdc) mutations demonstrate that the presence of the 120-kD isoform correlates with mitotic spindle for mation but not with SPB duplication. In a synchronous wild-type popula tion, the additional serine/threonine phosphorylation that gives rise to the 120-kD isoform appears as cells are forming the mitotic spindle and diminishes as cells enter anaphase. None of several sequences sim ilar to the consensus for phosphorylation by the Cdc28p (cdc2(p34)) ki nase is important for these mitosis-specific phosphorylations or for f unction. Carboxy-terminal Spc110p truncations lacking the calmodulin b inding site can support growth and are also phosphorylated in a cell c ycle-specific manner. Further truncation of the Spc110p carboxy termin us results in mutant proteins that are unable to support growth and no w migrate as single species. Collectively, these results provide the f irst evidence of a structural component of the SPB that is phosphoryla ted during spindle formation and dephosphorylated as cells enter anaph ase.