Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic C-terminal domain, termed the polo box. Here we show that the budding-yeast polo kinase Cdc5, when fused to green fluorescent protein and expressed under its endogenous promoter, localizes at spindle poles and the mother bud neck. Overexpression of Cdc5 can induce a class of cells with abnormally elongated buds in a polo box-and kinase activity-dependent manner. In addition to localizing at the spindle poles and cytokinetic neck filaments, Cdc5 induces and localizes to additional septin ring structures within the elongated buds. Without impairing kinase activity, conservative mutations in the polo box abolish the ability of Cdc5 to functionally complement the defect associated with a cdc5-1 temperature-sensitive mutation, to localize to the spindle poles and cytokinetic neck filaments, and to induce elongated cells with ectopic septin ring structures. Consistent with the polo box-dependent subcellular localization, the C-terminal domain of Cdc5, but not its polo box mutant, is sufficient for subcellular localization, and its overexpression appears to inhibit cytokinesis. These data provide evidence that the polo box is required to direct Cdc5 to specific subcellular locations and induce or organize cytokinetic structures.

The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters P A and P F , respectively. However, there is an additional weak promoter, P R , which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the P A and P F promoters were activated by D-xylose and repressed by glucose. In contrast, the P R promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the P A and P F promoters, causing a defect in both growth and transport.

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In eukaryotes, entry into mitosis is induced by cyclin Bbound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Here, we show that Hsl1 and Hsl7 are required for proper localization of Cdc5 (Pololike kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this modification served as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. We propose that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events.

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7⌬ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7⌬ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.Cytokinesis in animal cells involves an actomyosin-based contractile ring, which forms late in the cell cycle and constricts the plasma membrane, resulting in the division of one cell into two cells. In the budding yeast Saccharomyces cerevisiae, the cleavage plane is specified early in the cell cycle, and cytokinesis involves the concerted action of actomyosin ring contraction and septum formation (5, 37, 55). Despite some differences in the morphological features and timing of certain cytokinetic events between yeast and animal cells, many of the components involved are conserved (18). In addition to the conserved involvement of the actomyosin contractile ring, it is now widely appreciated that the septins also play a critical role in cytokinesis in both yeast and animal cells.Septins are a family of proteins that were identified first in yeast and subsequently in various other fungi and animals (for review, see references 20, 32, and 39). Septin family members possess at least 26% amino acid sequence identity. All of the known septins possess an N-terminal P-loop and other sequences conserved in the GTPase superfamily of nucleotidebinding proteins (8). In addition, at or near their C termini, most septins possess a predicted coiled-coil domain (for review, see reference 39) that may be important in proteinprotein interactions.In yeast, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p (or Shs1p) septins all localize to the presumptive bud site before bud emergence and remain at the mother-bud neck until after cytokinesis (11,21,28,33,45; M. S. Longtine, unpublished observations). Recent studies...