P63(CDC13), A B-TYPE CYCLIN, IS ASSOCIATED WITH BOTH THE NUCLEOLAR AND CHROMATIN DOMAINS OF THE FISSION YEAST NUCLEUS

The cellular distribution of the fission yeast mitotic cyclin B, p63(c dc13), was investigated by a combination of indirect immunofluorescenc e light microscopy, immunogold electron microscopy, and nuclear isolat ion and fractionation. Immunofluorescence microscopy of wild-type cell s and the cold-sensitive mutant dis2.11 with a monospecific anti-p63(c dc13) antiserum was consistent with the association of a major subpopu lation of fission yeast M-phase protein kinase with the nucleolus. Imm unogold electron microscopy of freeze-substituted wild-type cells iden tified two nuclear populations of p63(cdc13), one associated with the nucleolus, the other with the chromatin domain. To investigate the cel l cycle regulation of nuclear labeling, the mutant cdc25.22 was synchr onized through mitosis by temperature arrest and release. Immunogold l abeling of cells arrested at G(2)M revealed gold particles present abu ndantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63(cdc13), but few gold particles were detected over the cytopl asm. Labeling of all cell compartments declined to zero through mitosi s. Cell fractionation confirmed that p63(cdc13) was substantially enri ched in both isolated nuclei and in a fraction containing small vesicl es and organelles. p63(cdc13) was not extracted from nuclei by treatme nt with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, al though partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63 (cdc13), as did p34(cdc2), the other subunit of the M-phase protein ki nase. We conclude that a major subpopulation of the fission yeast mito tic cyclin B is targeted to structural elements of the nucleus and nuc leolus.