M. Andrew Hoyt

The steps in cell division are precisely orchestrated. A key checkpoint is inactivated after it has performed its function so that the next steps can take place.

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.

Previous studies of the spindle checkpoint suggested that its ability to prevent entry into anaphase was mediated by the inhibition of the anaphase-promoting complex (APC) ubiquitin ligase by Mad2. Two new studies challenge that view by demonstrating that another checkpoint protein, BubR1, is a far more potent inhibitor of APC function.

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.

Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly actingBUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G₂ DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, includingBUB2 and BUB3 and MAD1,MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.

We identified an allele of Saccharomyces cerevisiae CDC20 that exhibits a spindle-assembly checkpoint defect. Previous studies indicated that loss of CDC20 function caused cell cycle arrest prior to the onset of anaphase. In contrast, CDC20-50 caused inappropriate cell cycle progression through M phase in the absence of mitotic spindle function. This effect of CDC20-50 was dominant over wild type and was eliminated by a second mutation causing loss of function, suggesting that it encodes an overactive form of Cdc20p. Overexpression of CDC20 was found to cause a similar checkpoint defect, causing bypass of the preanaphase arrest produced by either microtubule-depolymerizing compounds or MPS1 overexpression. CDC20 overexpression was also able to overcome the anaphase delay caused by high levels of the anaphase inhibitor Pds1p, but not a mutant form immune to anaphase-promoting complex- (APC-)mediated proteolysis. CDC20 overexpression was unable to promote anaphase in cells deficient in APC function. These findings suggest that Cdc20p is a limiting factor that promotes anaphase entry by antagonizing Pds1p. Cdc20p may promote the APC-dependent proteolytic degradation of Pds1p and other factors that act to inhibit cell cycle progression through mitosis.

Motility is a hallmark of life. Even cells that are incapable of active movement within their environment perform essential intracellular motility processes. A number of mechanisms have evolved to generate the mechanical forces required to drive biological motility. A particularly successful and ubiquitous mechanism of biological force production utilizes mechanochemical enzymes, or ''motor proteins.'' These enzymes convert chemical energy, typically in the form of adenosine triphosphate (ATP), into mechanical force. Many of the movements, both cellular and subcellular, of eukaryotic cells are generated by the activities of motor proteins that act upon rigid cytoskeletal fibers. Three superfamilies of cytoskeletal motor proteins have been recognized. Motors of the myosin superfamily act upon actin filaments to generate cell surface contractions and other morphological changes, vesicle motility, cytoplasmic streaming, and muscle cell contraction. Members of the dynein and kinesin microtubule-based motor superfamilies move vesicles and organelles within cells, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Members of each motor superfamily can be recognized by the presence of a unique and conserved amino acid sequence that forms the ATP-binding and force-producing domain of the molecule. These conserved ''motor domains'' are linked to a diverse array of polypeptide sequences that presumably impart functional specificity to the various motors. Each of these superfamilies is populated with numerous distinct members that perform different cellular roles. In recent years, molecular genetic techniques have revealed novel motor proteins more quickly than our ability to determine their functions. The talks in this session of the German-American Frontiers of Science meeting addressed the cellular roles of some of these motor proteins. Mitotic Motors of the Yeast Saccharomyces cerevisiae. The segregation of replicated chromosomes in eukaryotic cells is accomplished by the mitotic spindle, a microtubule-based, force-producing organelle. For mitotically dividing S. cerevisiae (budding yeast) cells, the only essential role of microtubules appears to be for spindle function. Not surprisingly, all but one of the seven microtubule-based motors (one dynein and six kinesin-related) encoded by the S. cerevisiae genome have been linked to spindle function (Table 1). The exception, Smy1p, performs a nonessential role in polarized cell growth (1, 2). Due to functional overlap or redundancy, none of these seven motors are individually essential for cell viability. In this simple organism, every essential spindle movement is accomplished by more than one motor. The mitotic spindle is a bipolar structure composed of two half-spindles that face each other with their microtubules interdigitating. Bipolar spindle assembly requires the actions of kinesin-related proteins of the BimC family (3, 4). The two BimC motors in S. cerevisiae, Cin8p and Kip1p, also produce

The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1Δ, cin2Δ and cin4Δ. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant combinations are viable and no more affected than any single mutant. In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2Δ, cin2Δ and cin4Δ, but not that caused by other mutations that affect microtubule function. Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of β-tubulin into an assembly-competent state. Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.