Life is Degrading: SCF Ubiquitin Ligases -- Their Components and Functions

Author: Feldman, Reid Martin Renny

Year: 2000

Degree: Dissertation (Ph.D.)

Advisor: Deshaies, Raymond Joseph

Committee Members: Deshaies, Raymond Joseph; Bjorkman, Pamela J.; Campbell, Judith L.; Dunphy, William G.; Varshavsky, Alexander J.

Option: Biology

DOI: 10.7907/syk9-cn98

Abstract

The cell cycle is defined by a series of phases demarcated by biochemically imposed transitions. In the budding yeast S. cerevisiae, initial genetic experiments indicated that the G1-S phase transition requires the activities of the essential CDC4, CDC34, CDC53, and SKP1 gene products as well as an active G1 cyclin/Cdc28 kinase. These proteins promote entry into S phase by enabling the destruction of the S phase cyclin/Cdc28 inhibitor Sic1. That the ubiquitin proteolytic pathway plays a specific role in the elimination of Sic1 was suggested by the previous identification of Cdc34 as a ubiquitin conjugating, or E2, enzyme. However, the roles of the other gene products in eliminating Sic1 were not understood. We showed that Cdc4, Cdc53, and Skp1 assemble into a ubiquitin ligase complex named SCFCdc4. When combined with purified forms of Cdc34, the Uba1 ubiquitin-activating enzyme, and ubiquitin, SCFCdc4 catalyzes the in vitro ubiquitination of Sic1 molecules that were first phosphorylated by active cyclin/Cdc28. SCFCdc4 has emerged as a paradigm for elucidating the activities of highly evolutionarily conserved SCF complexes throughout eukaryotes. Within SCF, the F-box containing protein Cdc4 and its homologs act as receptors for phosphorylated substrates and are linked to the rest of SCF via F-box mediated binding to Skp1. Skp1 also interacts with Cdc53 or a related cullin family member. Cdc53 plus a newly identified SCF component, the essential RING-H2 protein Hrt1, form a highly conserved core ubiquitin ligase machine; their closest budding yeast homologs are present in the other known cell cycle ubiquitin ligase, the Anaphase Promoting Complex (APC). Cdc53/Hrt1 function by a novel mechanism to stimulate E2 enzyme activity, possibly by promoting an allosteric activation of E2 enzyme. The growing list of SCF components--including over 400 currently identified F-box proteins--and their substrates highlight the deep evolutionary conservation of SCF complexes and the diverse array of cellular processes that they control.

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