Clb5-associated Kinase Activity is Required Early in the Spindle Pathway for Correct Preanaphase Nuclear Positioning in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, a single cyclin-dependent kinase, Cdc28, regulates both G1/S and G2/M phase transitions by associating with stage-specific cyclins. During progression through S phase and G2/M, Cdc28 is activated by the B-type cyclins Clb1–6. Because of functional redundancy, specific roles for individual Clbs have been difficult to assign. To help genetically define such roles, strains carrying a cdc28ts allele, combined with single CLB deletions were studied. We assumed that by limiting the activity of the kinase, these strains would be rendered more sensitive to loss of individual Clbs.By this approach, a novel phenotype associated with CLB5 mutation was observed. Homozygous cdc28-4ts clb5 diploids were inviable at room temperature. Cells were defective in spindle positioning, leading to migration of undivided nuclei into the bud. Occasionally, misplaced spindles were observed in cdc28-4 clb5 haploids; additional deletion of CLB6 caused full penetrance. Thus, CLB5 effects proper preanaphase spindle positioning, yet the requirement differs in haploids and diploids. The execution point for the defect corresponded to the time of Clb5-dependent kinase activation. Nevertheless, lethality of cdc28-4 clb5 diploids was not rescued by CLB2 or CLB4 overexpression, indicating a specificity of Clb5 function beyond temporality of expression.

Conservation and Function of a Potential Substrate-Binding Domain in the Yeast Clb5 B-Type Cyclin

“…Two cdc28-4 clb5 pGAL-CLB5/ URA3 strains from this cross were mated. As expected from the work of Segal and Reed (32), the resulting diploid was completely inviable on glucose medium, unlike the parental haploids (data not shown), and was completely rescued by the introduction of either CDC28 or CLB5 on low-copy-number plasmids (see below). A clb5::HIS3 pds1::URA3 cdc20::LEU2 ADE2::GALL-CDC20 strain was constructed in the W303 background by crossing a pds1 cdc20 GALL-CDC20 strain with a clb5::HIS3 strain (both provided by M. Shirayama), followed by tetrad analysis (the GALL promoter is an attenuated GAL promoter used in the CDC20 construct to reduce toxicity [34]).…”

Overexpression of B-type cyclins alters chromosomal segregation

“…Nevertheless, in vertebrate cells, cyclins B1 and B2, which both associate with cdc2/ CDK1, and whose levels rise during G2 and peak in mitosis, have probably distinct roles: in interphase, cyclin B1 is localized mostly to microtubules whereas cyclin B2 is found in the Golgi apparatus (Jackman et al, 1995); cyclin B1/CDK1 complex controls chromosome condensation, reorganization of microtubules, destruction of the nuclear membrane, Golgi apparatus fragmentation whereas cyclin B2/CDK1 seems to be only involved in the disassembly of the Golgi apparatus (Draviam et al, 2001); cyclin B1 degradation precedes that of cyclin B2 during metaphase/anaphase transition (Minshull et al, 1990;Ohsumi et al, 1994;Brandeis et al, 1998); in contrast to cyclin B1, cyclin B2 does not translocate from the cytoplasm to the nucleus at the beginning of prophase (Jackman et al, 1995); cyclin B1 is an essential gene since its deletion in mice results in embryonic lethality whereas cyclin B2 7/7 mice develop normally (Brandeis et al, 1998). Cyclin B2, but not cyclin B1, was recently shown to be essential for a proper bipolar spindle formation in frog (Kotani et al, 2001), which is reminiscent of the biological role of Clb5 in yeast (Segal et al, 1998). The present study shows that overexpression of human cyclin B2, like overexpression of Clb5, alters the spindle checkpoint and chromosome segregation.…”

“…Clb5 has been shown to be essential for a proper preanaphase spindle positioning (Segal et al, 1998). Clb5 is degraded just before the metaphase to anaphase transition by APC Cdc20 and its degradation is required for the mitosis exit (Shirayama et al, 1999).…”

Bud6 Directs Sequential Microtubule Interactions with the Bud Tip and Bud Neck during Spindle Morphogenesis inSaccharomyces cerevisiae

“…Interestingly, such loss of spindle polarity resulted in lethality solely in diploids and was tolerated by haploids. Of the genetically determined characteristics associated with diploid versus haploid cells, budding pattern Zahner et al, 1996) best correlated with determining lethality in this system (Segal et al, 1998). Haploid cdc28-4 clb5⌬ cells budding bipolarly (a/␣ diploid budding pattern) by virtue of a bud3 mutation (Chant and Herskowitz, 1991), displayed comparable lethality to that of cdc28-4 clb5⌬ diploids.…”