Key Concepts Of Section 214

Molecular Mechanisms for Regulating Mitotic Events

■ Early in mitosis, MPF-catalyzed phosphorylation of lamins A, B, and C and of nucleoporins and inner nuclear envelope proteins causes depolymerization of lamin filaments (see Figure 21-16) and dissociation of nuclear pores into pore subcomplexes, leading to disassembly of the nuclear envelope and its retraction into the ER.

■ Phosphorylation of condensin complexes by MPF or a kinase regulated by MPF promotes chromosome condensation early in mitosis.

■ Sister chromatids formed by DNA replication in the S phase are linked at the centromere by cohesin complexes that contain DNA-binding SMC proteins and other proteins.

■ At the onset of anaphase, the APC is directed by Cdc20 to polyubiquitinate securin, which subsequently is degraded by proteasomes. This activates separase, which cleaves a subunit of cohesin, thereby unlinking sister chro-matids (see Figure 21-19).

■ After sister chromatids have moved to the spindle poles, the APC is directed by Cdh1 to polyubiquitinate mitotic cyclins, leading to their destruction and causing the decrease in MPF activity that marks the onset of telophase.

■ The fall in MPF activity in telophase allows constitutive protein phosphatases to remove the regulatory phosphates from condensin, lamins, nucleoporins, and other nuclear membrane proteins, permitting the decondensation of chromosomes and the reassembly of the nuclear membrane, nuclear lamina, and nuclear pore complexes.

■ The association of Ran-GEF with chromatin results in a high local concentration of Ran • GTP near the decon-densing chromosomes, promoting the fusion of nuclear envelope extensions from the ER around each chromosome. This forms karyomeres that then fuse to form daughter cell nuclei (see Figure 21-20).

■ The fall in MPF activity also removes its inhibition of myosin light chain, allowing the cleavage furrow to form and cytokinesis to proceed.

In most vertebrate cells the key decision determining whether or not a cell will divide is the decision to enter the S phase. In most cases, once a vertebrate cell has become committed to entering the S phase, it does so a few hours later and progresses through the remainder of the cell cycle until it completes mitosis. sS. cerevisiae cells regulate their proliferation similarly, and much of our current understanding of the molecular mechanisms controlling entry into the S phase and the control of DNA replication comes from genetic studies of S. cerevisiae.

S. cerevisiae cells replicate by budding (Figure 21-21). Both mother and daughter cells remain in the G1 period of the cell cycle while growing, although it takes the initially larger mother cells a shorter time to reach a size compatible with cell division. When ,S. cerevisiae cells in G1 have grown sufficiently, they begin a program of gene expression that leads to entry into the S phase. If G1 cells are shifted from a rich medium to a medium low in nutrients before they reach a critical size, they remain in G1 and grow slowly until they are large enough to enter the S phase. However, once G1 cells reach the critical size, they become committed to completing the cell cycle, entering the S phase and proceeding through G2 and mitosis, even if they are shifted to a medium low in nutrients. The point in late G1 of growing ,S. cerevisiae cells when they become irrevocably committed to entering the S phase and traversing the cell cycle is called START. As we shall see in Section 21.6, a comparable phenomenon occurs in replicating mammalian cells.

A Cyclin-Dependent Kinase (CDK) Is Critical for S-Phase Entry in S. cerevisiae

All sS. cerevisiae cells carrying a mutation in a particular cdc gene arrest with the same size bud at the nonpermissive temperature (see Figure 9-6b). Each type of mutant has a terminal phenotype with a particular bud size: no bud (cdc28), intermediate-sized buds, or large buds (cdc7). Note that in S. cerevisiae wild-type genes are indicated in italic capital letters (e.g., CDC28) and recessive mutant genes in italic lowercase letters (e.g., cdc28); the corresponding wild-type

► FIGURE 21-21 The budding yeast S. cerevisiae.

(a) Scanning electron micrograph of S. cerevisiae cells at various stages of the cell cycle. The larger the bud, which emerges at the end of the G1 phase, the further along in the cycle the cell is. (b) Main events in S. cerevisiae cell cycle. Daughter cells are born smaller than mother cells and must grow to a greater extent in G1 before they are large enough to enter the S phase. As in S. pombe, the nuclear envelope does not break down during mitosis. Unlike S. pombe chromosomes, the small S. cerevisiae chromosomes do not condense sufficiently to be visible by light microscopy. [Part (a) courtesy of E. Schachtbach and I. Herskowitz.]

protein is written in Roman letters with an initial capital (e.g., Cdc28), similar to pombe proteins.

The phenotypic behavior of temperature-sensitive cdc28 mutants indicates that Cdc28 function is critical for entry into the S phase. When these mutants are shifted to the non-permissive temperature, they behave like wild-type cells suddenly deprived of nutrients. That is, cdc28 mutant cells that have grown large enough to pass START at the time of the temperature shift continue through the cell cycle normally and undergo mitosis, whereas those that are too small to have passed START when shifted to the nonpermissive temperature do not enter the S phase even though nutrients are plentiful. Even though cdc28 cells blocked in G1 continue to grow in size at the nonpermissive temperature, they cannot pass START and enter the S phase. Thus they appear as large cells with no bud.

The wild-type CDC28 gene was isolated by its ability to complement mutant cdc28 cells at the nonpermissive temperature (see Figure 21-4). Sequencing of CDC28 showed that the encoded protein is homologous to known protein kinases, and when Cdc28 protein was expressed in E. coli, it exhibited protein kinase activity. Actually, Cdc28 from sS. cerevisiae was the first cell-cycle protein shown to be a protein kinase. Subsequently, the wild-type ,S. pombe cdc2+ gene was found to be highly homologous to the ,S. cerevisiae CDC28 gene, and the two encoded proteins—Cdc2 and Cdc28—are functionally analogous. Each type of yeast contains a single cyclin-dependent protein kinase (CDK), which can substitute for each other: Cdc2 in ,S. pombe and Cdc28 in S. cerevisiae (see Table 21-1).

The difference in the mutant phenotypes of cdc2~ S. pombe cells and cdc28 S. cerevisiae cells can be explained in terms of the physiology of the two yeasts. In S. pombe cells growing in rich media, cell-cycle control is exerted primarily at the G2 ^ M transition (i.e., entry to mitosis). In many cdc2T mutants, including those isolated first, enough Cdc2 activity is maintained at the nonpermissive temperature to permit cells to enter the S phase, but not enough to permit entry into mitosis. Such mutant cells are observed to be elongated cells arrested in G2. At the nonpermissive temperature, cultures of completely defective cdc2~ mutants include some cells arrested in G1 and some arrested in G2, depending on their location in the cell cycle at the time of the temperature shift. Conversely, cell-cycle regulation in ,S. cerevisiae is exerted primarily at the G1 ^ S transition (i.e., entry to the S phase). Therefore, partially defective cdc28 cells are arrested in G1, but completely defective cdc28 cells are arrested in either G1 or G2. These observations demonstrate that both the sS. pombe and the ,S. cerevisiae CDKs are required for entry into both the S phase and mitosis.

Three Gi Cyclins Associate with S. cerevisiae CDK to form S Phase-Promoting Factors

By the late 1980s, it was clear that mitosis-promoting factor (MPF) is composed of two subunits: a CDK and a mitotic

B-type cyclin required to activate the catalytic subunit. By analogy, it seemed likely that .S. cerevisiae contains an S phase-promoting factor (SPF) that phosphorylates and regulates proteins required for DNA synthesis. Similar to MPF, SPF was proposed to be a heterodimer composed of the sS. cerevisiae CDK and a cyclin, in this case one that acts in G1 (see Figure 21-2, steps (2-0).

To identify this putative G1 cyclin, researchers looked for genes that, when expressed at high concentration, could suppress certain temperature-sensitive mutations in the .S. cerevisiae CDK. The rationale of this approach is illustrated in Figure 21-22. Researchers isolated two such genes designated CLN1 and CLN2. Using a different approach, researchers identified a dominant mutation in a third gene called CLN3. Sequencing of the three CLN genes showed that they encoded related proteins each of which includes an «100-residue region exhibiting significant homology with B-type cyclins from sea urchin, Xenopus, human, and sS. pombe. This region encodes the cyclin domain that interacts with CDKs and is included in the domain of human cy-clin A shown in Figure 21-15b,c. The finding that the three Cln proteins contain this region of homology with mitotic cyclins suggested that they were the sought-after .S. cerevisiae G1 cyclins. (Note that the homologous CDK-binding domain found in various cyclins differs from the destruction box mentioned earlier, which is found only in B-type cyclins.)

Gene knockout experiments showed that .S. cerevisiae cells can grow in rich medium if they carry any one of the three G1 cyclin genes. As the data presented in Figure 21-23 indicate, overproduction of one G1 cyclin decreases the fraction of cells in G1, demonstrating that high levels of the G1 cyclin-CDK complex drive cells through START prematurely. Moreover, in the absence of any of the G1 cyclins, cells become arrested in G1, indicating that a G1 cyclin-CDK het-erodimer, or SPF, is required for .S. cerevisiae cells to enter the S phase. These findings are reminiscent of the results for the sS. pombe mitotic cyclin (Cdc13) with regard to passage through G2 and entry into mitosis. Overproduction of the mitotic cyclin caused a shortened G2 and premature entry into mitosis, whereas inhibition of the mitotic cyclin by mutation resulted in a lengthened G2 (see Figure 21-12). Thus these results confirmed that the .S. cerevisiae Cln proteins are G1 cyclins that regulate passage through the G1 phase of the cell cycle.

In wild-type yeast cells, CLN3 mRNA is produced at a nearly constant level throughout the cell cycle, but its translation is regulated in response to nutrient levels. The CLN3 mRNA contains a short upstream open-reading frame that inhibits initiation of translation of this mRNA. This inhibition is diminished when nutrients and hence translation initiation factors are in abundance. Since Cln3 is a highly unstable protein, its concentration fluctuates with the translation rate of CLN3 mRNA. Consequently, the amount and activity of Cln3-CDK complexes, which depends on the concentration of Cln3 protein, is largely regulated by the nutrient level.

(a)

25 °C

cyclin^

36 °C

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