Info

cdc28ts cells

Wild-type G, cyclin

Mutant, low-affinity CDK

Colonies form Arrested in G,

Wild-type G, cyclin

▲ EXPERIMENTAL FIGURE 21-22 Genes encoding two S. cerevisiae G1 cyclins were identified by their ability to supress a temperature-sensitive mutant CDK. This genetic screen is based on differences in the interactions between Gt cyclins and wild-type and temperature-sensitive (ts) S. cerevisiae CDKs. (a) Wild-type cells produce a normal CDK that associates with Gt cyclins, forming the active S phase-promoting factor (SPF), at both the permissive and nonpermissive temperature (i.e., 25 ° and 36 °C). (b) Some cdc28ts mutants express a mutant CDK with low affinity for Gt cyclin at 36 °C. These mutants produce enough Gt cyclin-CDK (SPF) to support growth and colony development at 25 °C, but not at 36 °C. (c) When cdc28ts cells were transformed with a S. cerevisiae genomic library cloned in a high-copy plasmid, three types of colonies formed at 36 °C: one contained a plasmid carrying the wild-type CDC28 gene; the other two contained plasmids carrying either the CLN1 or CLN2 gene. In transformed cells carrying the CLN1 or CLN2 gene, the concentration of the encoded Gt cyclin is high enough to offset the low affinity of the mutant CDK for a Gt cyclin at 36 °C, so that enough SPF forms to support entry into the S phase and subsequent mitosis. Untransformed cdc28ts cells and cells transformed with plasmids carrying other genes are arrested in Gt and do not form colonies. [See J. A. Hadwiger et al., 1989, Proc. Nat'l. Acad. Sci. USA 86:6255.]

Cln3 expression vector

GAL1 G-| cyclin gene promoter

- Glucose

High-level expression of G1 cyclin

- Glucos^^^ No G1 cyclin exPression

(a) Wild-type cells + empty vector e o

(a) Wild-type cells + empty vector e o

G1 G2 Fluorescence -

(b) Wild-type cells + G1 cyclin vector e o

G1 G2 Fluorescence -

g1 g2 Fluorescence

(c) clnl Icln2 Icln3 cells + G1 cyclin vector

(b) Wild-type cells + G1 cyclin vector e o

g1 g2 Fluorescence e o

(c) clnl Icln2 Icln3 cells + G1 cyclin vector e o

G1 G2 Fluorescence

▲ EXPERIMENTAL FIGURE 21-23 Overexpression of G1 cyclin prematurely drives S. cerevisiae cells into the S phase.

The yeast expression vector used in these experiments (top) carried one of the three S. cerevisiae G1 cyclin genes linked to the strong GAL1 promoter, which is turned off when glucose is present in the medium. To determine the proportion of cells in G1 and G2, cells were exposed to a fluorescent dye that binds to DNA and then were passed through a fluorescence-activated cell sorter (see Figure 5-34). Since the DNA content of G2 cells is twice that of G1 cells, this procedure can distinguish cells in the two cell-cycle phases. (a) Wild-type cells transformed with an empty expression vector displayed the normal distribution of cells in G1 and G2 in the absence of glucose (Glc) and after addition of glucose. (b) In the absence of glucose, wild-type cells transformed with the G1 cyclin expression vector displayed a higher-than-normal percentage of cells in the S phase and G2 because overexpression of the G1 cyclin decreased the G1 period (top curve). When expression of the G1 cyclin from the vector was shut off by addition of glucose, the cell distribution returned to normal (bottom curve). (c) Cells with mutations in all three G1 cyclin genes and transformed with the G1 cyclin expression vector also showed a high percentage of cells in S and G2 in the absence of glucose (top curve). Moreover, when expression of G1 cyclin from the vector was shut off by addition of glucose, the cells completed the cell cycle and arrested in G1 (bottom curve), indicating that a G1 cyclin is required for entry into the S phase. [Adapted from H. E. Richardson et al., 1989, Cell 59:1127.]

Once sufficient Cln3 is synthesized from its mRNA, the Cln3-CDK complex phosphorylates and activates two related transcription factors, SBF and MBF. These induce transcription of the CLN1 and CLN2 genes whose encoded proteins accelerate entry into the S phase. Thus regulation of CLN3 mRNA translation in response to the concentration of nutrients in the medium is thought to be primarily responsible for controlling the length of G1 in cerevisiae. SBF and MBF also stimulate transcription of several other genes required for DNA replication, including genes encoding DNA polymerase subunits, RPA subunits (the eukaryotic ssDNA-binding protein), DNA ligase, and enzymes required for deoxyribonucleotide synthesis.

One of the important substrates of the late G1 cyclin-CDK complexes (Clnl-CDK and Cln2-CDK in S. cerevisiae) is Cdhl. Recall that this specificity factor directs the APC to polyubiquitinate B-type cyclins during late anaphase of the previous mitosis, marking these cyclins for proteolysis by proteasomes (see Figure 21-10). The MBF transcription factor activated by the Cln3-CDK complex also stimulates transcription of CLB5 and CLB6, which encode cyclins of the B-type, hence their name. Because the complexes formed between these B-type cyclins and the ,S. cerevisiae CDK are required for initiation of DNA synthesis, the Clb5 and Clb6 proteins are called S-phase cyclins. Inactivation of the APC earlier in G1 allows the S-phase cyclin-CDK complexes to accumulate in late G1. The specificity factor Cdh1 is phos-phorylated and inactivated by both late G1 and B-type cyclin-CDK complexes, and thus remains inhibited throughout S, G2, and M phase until late anaphase.

Degradation of the S-Phase Inhibitor Triggers DNA Replication

As the S-phase cyclin-CDK heterodimers accumulate in late G1, they are immediately inactivated by binding of an inhibitor, called ,Sic1, that is expressed late in mitosis and in early G1. Because Sic1 specifically inhibits B-type cyclin-CDK complexes, but has no effect on the G1 cyclin-CDK complexes, it functions as an S-phase inhibitor.

Entry into the S phase is defined by the initiation of DNA replication. In ,S. cerevisiae cells this occurs when the Sic1 inhibitor is precipitously degraded following its polyubiqui-tination by a distinct ubiquitin ligase called SCF (Figure 21-24; see also Figure 21-2, step 5). Once Sic1 is degraded, the S-phase cyclin-CDK complexes induce DNA replication by phosphorylating multiple proteins bound to replication origins. This mechanism for activating the S-phase cyclin-CDK com-plexes—that is, inhibiting them as the cyclins are synthesized and then precipitously degrading the inhibitor—permits the sudden activation of large numbers of complexes, as opposed to the gradual increase in kinase activity that would result if no inhibitor were present during synthesis of the S-phase cyclins.

We can now see that regulated proteasomal degradation directed by two ubiquitin ligase complexes, SCF and APC, controls three major transitions in the cell cycle: onset of the S phase through degradation of Sic1, the beginning of anaphase through degradation of securin, and exit from mitosis through degradation of B-type cyclins. The APC is directed to polyubiquitinate securin, which functions as an anaphase inhibitor, by the Cdc20 specificity factor (see Figure 21-19). Another specificity factor, Cdh1, targets APC to B-type cyclins (see Figure 21-10). The SCF is directed to polyubiquitinate Sic1 by a different mechanism, namely, phosphorylation of Sic1 by a G1 cyclin-CDK (see Figure 21-24). This difference in strategy probably occurs because the APC has several substrates, including securin and B-type cyclins, which must be degraded at different times in the cycle. In contrast, entry into the S phase requires the degradation of only a single protein, the Sic1 inhibitor. An obvious advantage of proteolysis for controlling passage through these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle.

Multiple Cyclins Direct the Kinase Activity of S. cerevisiae CDK During Different Cell-Cycle Phases

As budding yeast cells progress through the S phase, they begin transcribing genes encoding two additional B-type cy-clins, Clb3 and Clb4. These form heterodimeric cyclin-CDK complexes that, together with complexes including Clb5 and Clb6, activate DNA replication origins throughout the remainder of the S phase. The Clb3-CDK and Clb4-CDK complexes also initiate formation of the mitotic spindle at the beginning of mitosis. When ,S. cerevisiae cells complete chromosome replication and enter G2, they begin expressing two more B-type cyclins, Clb1 and Clb2. These function as mitotic cyclins, associating with the CDK to form complexes that are required for mediating the events of mitosis.

Each group of cyclins thus directs the ,S. cerevisiae CDK to specific functions associated with various cell-cycle phases, as outlined in Figure 21-25. Cln3-CDK induces expression of Cln1, Cln2, and other proteins in mid-late G1 by phosphorylating and activating the SBF and MBF transcription factors. Cln1-CDK and Cln2-CDK inhibit the APC, allowing B-type cyclins to accumulate; these G1 cyclin-CDKs also activate degradation of the S-phase inhibitor Sic1. The S-phase CDK complexes containing Clb5, Clb6, Clb3, and Clb4 then trigger DNA synthesis. Clb3 and Clb4 also function as mitotic

Polyubiquitination; proteasomal degradation

Mid-late G

Mid-late G

Polyubiquitination; proteasomal degradation

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