We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures sync...

Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.

A chemical switch for inhibitor-sensitive alleles of any protein kinase

Proteolysis mediated by the anaphase-promoting complex (APC) triggers chromosome segregation and exit from mitosis, yet its regulation is poorly understood. The conserved Cdc20 and Cdh1 proteins were identified as limiting, substrate-specific activators of APC-dependent proteolysis. CDC20 was required for the degradation of the APC substrate Pds1 but not for that of other APC substrates, such as Clb2 and Ase1. Conversely, cdh1Δmutants were impaired in the degradation of Ase1 and Clb2 but not in that of Pds1. Overexpression of either CDC20 orCDH1 was sufficient to induce APC-dependent proteolysis of the appropriate target in stages of the cell cycle in which substrates are normally stable.

https://science.sciencemag.org/content/sci/278/5337/460.full.pdf

CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis.

The ubiquitin/proteasome system is believed to be the major nonlysosomal proteolytic system of eukaryotic cells. It is present in the cytosol and the nucleus but apparently absent from the lumen of membrane-enclosed organelles, i. e., the endoplasmic reticulum, the Golgi and vesicular system, mitochondria, chloroplasts, and peroxisomes (for reviews see Finley and Chau, 1991; Jentsch, 1992a,b; Hershko and Ciechanover, 1992; Ciechanover, 1994; Hochstrasser, 1995; Smith et al., 1996). Substrates of this pathway include soluble proteins, subunits of oligomeric protein complexes, and integral membrane proteins. An important function of this pathway is the elimination of abnormal proteins (e. g., misfolded, misassembled) generated under normal and, in particular, stress conditions. Moreover, it is assumed that most naturally short-lived proteins of the cytosol and the nucleus are degraded by this pathway. Known substrates include proteins with important regulatory roles such as transcription factors, cell cycle regulators, and signal transducers.

The Ubiquitin-Conjugation System

In budding yeast, multiple cyclins activate a single cyclin-dependent kinase to control progression through the cell cycle. Different mechanisms of cyclin specificity are thought to be important for the correct order and timing of cell-cycle events. Cyclins regulate the cell cycle by binding to and activating cyclin-dependent kinases (Cdks). Phosphorylation of specific targets by cyclin–Cdk complexes sets in motion different processes that drive the cell cycle in a timely manner. In budding yeast, a single Cdk is activated by multiple cyclins. The ability of these cyclins to target specific proteins and to initiate different cell-cycle events might, in some cases, reflect the timing of the expression of the cyclins; in others, it might reflect intrinsic properties of the cyclins that render them better suited to target particular proteins.

/pdf/multiple-levels-of-cyclin-specificity-in-cell-cycle-control-2hf27nouhk.pdf

Multiple levels of cyclin specificity in cell-cycle control.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.

L Goetsch

Papers

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.