▲ FIGURE 21-2 Overview of current model for regulation of the eukaryotic cell cycle. Passage through the cycle is controlled by G1, S-phase, and mitotic cyclin-dependent kinase complexes (green). These are composed of a regulatory cyclin subunit and a catalytic cyclin-dependent kinase (CDK) subunit. Two ubiquitin ligase complexes (orange), SCF and APC, polyubiquitinate specific substrates including S-phase inhibitors (step 5), securin (step 8), and mitotic cyclins (step 9), marking these substrates for degradation by proteasomes. Proteolysis of the S-phase inhibitor activates S-phase cyclin-CDK complexes, leading to chromosome replication. Proteolysis of securin results in degradation of protein complexes that connect sister chromatids at metaphase, thereby initiating anaphase, the mitotic period in which sister chromatids are separated and moved to the opposite spindle poles. Reduction in the activity of mitotic cyclin-CDK complexes caused by proteolysis of mitotic cyclins permits late mitotic events and cytokinesis to occur. These proteolytic cleavages drive the cycle in one direction because of the irreversibility of protein degradation. See text for further discussion.

transcription of genes encoding enzymes required for DNA synthesis and the genes encoding S-phase cyclins and CDKs. The activity of S-phase cyclin-CDK complexes is initially held in check by inhibitors. Late in Gj, the G1 cyclin-CDK complexes induce degradation of the S-phase inhibitors by phos-phorylating them and consequently stimulating their polyubiquitination by the multiprotein SCF ubiquitin ligase (step 5). Subsequent degradation of the polyubiquitinated S-phase inhibitor by proteasomes releases active S-phase cyclin-CDK complexes.

Once activated, the S-phase cyclin-CDK complexes phosphorylate regulatory sites in the proteins that form DNA pre-replication complexes, which are assembled on replication origins during G1 (Chapter 4). Phosphorylation of these proteins not only activates initiation of DNA replication but also prevents reassembly of new pre-replication complexes. Because of this inhibition, each chromosome is replicated just once during passage through the cell cycle, ensuring that the proper chromosome number is maintained in the daughter cells.

Mitotic cyclin-CDK complexes are synthesized during the S phase and G2, but their activities are held in check by phosphorylation at inhibitory sites until DNA synthesis is completed. Once activated by dephosphorylation of the inhibitory sites, mitotic cyclin-CDK complexes phosphorylate multiple proteins that promote chromosome condensation, retraction of the nuclear envelope, assembly of the mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate. During mitosis, the anaphase-promoting complex (APC), a multisubunit ubiquitin ligase, polyubiquitinates key regulatory proteins marking them for proteasomal degradation. One important substrate of the APC is securin, a protein that inhibits degradation of the cross-linking proteins between sister chromatids. The poly-ubiquitination of securin by the APC is inhibited until the kinetochores assembled at the centromeres of all chromosomes have become attached to spindle microtubules, causing chromosomes to align at the metaphase plate. Once all the chromosomes are aligned, the APC polyubiquitinates se-curin, leading to its proteasomal degradation and the subsequent degradation of the cross-linking proteins connecting sister chromatids (see Figure 21-2, step 8). This sequence of events initiates anaphase by freeing sister chromatids to segregate to opposite spindle poles.

Late in anaphase, the APC also directs polyubiquitina-tion and subsequent proteasomal degradation of the mitotic cyclins. Polyubiquitination of the mitotic cyclins by APC is inhibited until the segregating chromosomes have reached the proper location in the dividing cell (see Figure 21-2, step 9). Degradation of the mitotic cyclins leads to inactivation of the protein kinase activity of the mitotic CDKs. The resulting decrease in mitotic CDK activity permits constitu-tively active protein phosphatases to remove the phosphates that were added to specific proteins by the mitotic cyclin-CDK complexes. As a result, the now separated chromosomes decondense, the nuclear envelope re-forms around daughter-cell nuclei, and the Golgi apparatus reassembles during telophase; finally, the cytoplasm divides at cytokinesis, yielding the two daughter cells.

During early G1 of the next cell cycle, phosphatases de-phosphorylate the proteins that form pre-replication complexes. These proteins had been phosphorylated by S-phase cyclin-CDK complexes during the previous S phase, and their phosphorylation was maintained during mitosis by mitotic cyclin-CDK complexes. As a result of their dephosphoryla-tion in G1, new pre-replication complexes are able to reassemble at replication origins in preparation for the next S phase (see Figure 21-2, step 1). Phosphorylation of Cdhl by G1 cyclin-CDK complexes in late G1 inactivates it, allowing accumulation of S-phase and mitotic cyclins during the ensuing cycle.

Passage through three critical cell-cycle transitions— G1 ^ S phase, metaphase ^ anaphase, and anaphase ^ telophase and cytokinesis—is irreversible because these transitions are triggered by the regulated degradation of proteins, an irreversible process. As a consequence, cells are forced to traverse the cell cycle in one direction only.

In higher organisms, control of the cell cycle is achieved primarily by regulating the synthesis and activity of G1 cyclin-CDK complexes. Extracellular growth factors function as mitogens by inducing synthesis of G1 cyclin-CDK complexes. The activity of these and other cyclin-CDK complexes is regulated by phosphorylation at specific inhibitory and activating sites in the catalytic subunit. Once mitogens have acted for a sufficient period, the cell cycle continues through mitosis even when they are removed. The point in late G1 where passage through the cell cycle becomes independent of mitogens is called the restriction point (see Figure 21-2).

Diverse Experimental Systems Have Been Used to Identify and Isolate Cell-Cycle Control Proteins

The first evidence that diffusible factors regulate the cell cycle came from cell-fusion experiments with cultured mammalian cells. When interphase cells in the G1, S, or G2 phase of the cell cycle were fused to cells in mitosis, their nuclear envelopes retracted and their chromosomes condensed (Figure 21-3). This finding indicates that some diffusible component or components in the cytoplasm of the mitotic cells forced interphase nuclei to undergo many of the processes associated with early mitosis. We now know that these factors are the mitotic cyclin-CDK complexes.

Similarly, when cells in G1 were fused to cells in S phase and the fused cells exposed to radiolabeled thymidine, the label was incorporated into the DNA of the G1 nucleus as well as the S-phase nucleus, indicating that DNA synthesis began in the G1 nucleus shortly after fusion. However, when cells in G2 were fused to S-phase cells, no incorporation of labeled thymidine occurred in the G2 nuclei. Thus diffusible factors in an S-phase cell can enter the nucleus of a G1 cell and stimulate DNA synthesis, but these factors cannot in-

However, complementation of the recessive mutation by the wild-type allele carried by one of the plasmid clones in the library allows a transformed mutant cell to grow into a colony; the plasmids bearing the wild-type allele can then be recovered from those cells. Because many of the proteins that regulate the cell cycle are highly conserved, human cDNAs cloned into yeast expression vectors often can complement yeast cell-cycle mutants, leading to the rapid isolation of human genes encoding cell-cycle control proteins.

Biochemical studies require the preparation of cell extracts from many cells. For biochemical studies of the cell cycle, the eggs and early embryos of amphibians and marine invertebrates are particularly suitable. In these organisms, multiple synchronous cell cycles follow fertilization of a large egg. By isolating large numbers of eggs from females and

▲ EXPERIMENTAL FIGURE 21-3 A diffusable factor in mitotic cells can induce mitosis in an interphase cell. In unfused interphase cells, the nuclear envelope is intact and the chromosomes are not condensed, so individual chromosomes cannot be distinguished (see Figures 1 -2b and 5-25). In mitotic cells, the nuclear envelope is absent and the individual replicated chromosomes are highly condensed. This micrograph shows a hybrid cell resulting from fusion of a mitotic cell (left side) with an interphase cell in G1 (right side). A factor from the mitotic cell cytoplasm has caused the nuclear envelope of the G1 cell to retract into the endoplasmic reticulum, so that it is not visible. The factor has also caused the G1 cell chromosomes to partially condense. The mitotic chromosomes can be distinguished because the two sister chromatids are joined at the centromere. [From R. T Johnson and P N. Rao, 1970, Biol. Rev. 46:97.]

duce DNA synthesis in a G2 nucleus. We now know that these factors are S-phase cyclin-CDK complexes, which can activate the pre-replication complexes assembled on DNA replication origins in early G1 nuclei. Although these cellfusion experiments demonstrated that diffusible factors control entry into the S and M phases of the cell cycle, genetic and biochemical experiments were needed to identify these factors.

The budding yeast Saccharomyces cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe have been especially useful for isolation of mutants that are blocked at specific steps in the cell cycle or that exhibit altered regulation of the cycle. In both of these yeasts, temperature-sensitive mutants with defects in specific proteins required to progress through the cell cycle are readily recognized microscopically and therefore easily isolated (see Figure 9-6). Such cells are called cdc (cell-division cycle) mutants. The wild-type alleles of recessive temperature-sensitive cdc mutant alleles can be isolated readily by transforming haploid mutant cells with a plasmid library prepared from wild-type cells and then plating the transformed cells at the nonpermissive temperature (Figure 21-4). When plated out, the haploid mutant cells cannot form colonies at the nonpermissive temperature.

cdc28is cells grown at 25 °C

Transform with plasmid library of wild-type S. cerevisiae DNA

Gene X

Gene Y

Transformed cdc28ts cells grown at 35 °C

, No colony Í formation

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