Milestone 22

Branded for breakdown Breaking up is hard to do — but breaking down is key to getting through the cell cycle. The discovery of the anaphase-promoting complex (APC) — a ligase that, together with a ubiquitin-activating enzyme and a ubiqutin- conjugating enzyme, targets proteins for degradation — opened up the field of proteolysis (see Milestone 20). The next exciting instalment came in 1994 when Kim Nasmyth and his colleagues, working on the budding yeast , showed that proteolysis is also required for cells to begin DNA replication. The responsible for this was subsequently identified as SCF.

Unlike the APC, SCF specifically recognizes proteins that are phosphorylated. So the SCF provides a link between regulators that are involved in and proteolysis, piecing together a central regulatory pathway in the eukaryotic cell cycle. It was known that an active -dependent kinase, in this case Cdc28, is required for passage through the G1 to S checkpoint of the cell cycle, and that Cdc28 can be activated by the G1 , Cln1, 2 and 3. But that's only half the story, as Nasmyth and his team showed. DNA replication also depends on activation of Cdc28 by B-type cyclins (Clbs); mutants lacking all six Clb proteins fail to enter but keep on budding. This resembles mutants in Cdc34, a ubiquitin-conjugating enzyme. Cdc34 might be needed to break down an inhibitor of Clb-Cdc28 kinases, the team reasoned. They successfully pinpointed the key inhibitor Sic1, which keeps Clb- Cdc28 complexes inactive; proteolysis of Sic1 releases the inhibition, allowing DNA replication to get underway. Degradation of specific proteins came to be recognized as crucial for progression through three cell-cycle transitions: S-phase entry, separation of sister chromatids and exit from mitosis. Subsequent work teased apart the complex of proteins that ubiquitylate, and so mark for breakdown, Sic1 and other inhibitory proteins. Stephen Elledge and his colleagues showed that the SKP1 gene is evolutionarily conserved, and involved in linking ubiquitylated proteins to proteolysis during S- phase entry. Importantly, they showed that Skp1 binds to a number of different proteins that contain a novel motif — the F-box. These F-box proteins are responsible for binding to phosphorylated substrates, such as Sic1. A database search showed that there are more than 40 different F-box proteins, and introduced the possibility that there could be several types of Skp-F-box complexes, all with different substrate specificities.

Subsequent work established that Skp1 forms a central component of a newly discovered ubiquitin ligase. By in vitro reconstruction of the Sic1 ubiquitylation pathway, Elledge's and Raymond Deshaies' groups showed that Skp1 assembles a ubiquitin ligase complex called SCF that also contains a protein of the cullin family, such as Cdc53, and an F-box protein. Together with Cdc34, one particular SCF complex, composed of Skp1, Cdc53 and Cdc4, recognizes and degrades Sic1 — but only once it is phosphorylated by the Cln-Cdc28 complex. So the teams dissected how phosphorylation and proteolysis link together to regulate the cell cycle.

Helen Pearson, Science Writer, Nature References

ORIGINAL RESEARCH PAPER Schwob, E. et al. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell 79, 233-244 (1994) Bai, C. et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, 263-274 (1996) Skowyra, D. et al. F-Box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91, 209-219 (1997) Feldman, R. M. R. et al. A complex of Cdc4p, Skp1p and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell 91, 221-230 (1997)

FURTHER READING Goebl, M. G. et al. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science 241, 1331-1335 (1988)