Cdc4p defines a family of evolutionarily conserved
proteins found in fungi, plants and mammals. In yeast, Cdc4p is a
component of an ubiquitin (Ub) ligase complex that is required for the
transfer of Ub onto key cell cycle regulators, thus targeting those
regulators for degradation. To define functional domains on Cdc4p a
panel of GST-Cdc4p fusion proteins was produced. We have defined two
domains on Cdc4p involved in protein-protein interactions. The F-box, a
conserved stretch of 45 amino acids, is sufficient to bind Cdc53p though
Skp1p; two other components of the E3 ligase complex. The WD-40 repeats
are sufficient to bind Cdc34p, a Ub conjugating enzyme. We have
identified two domains on Cdc4p that regulate Cdc4p activity. The N-terminus of Cdc4p is dispensable for Cdc4p activity yet when
overproduced in cells generates a cell cycle arrest consistent with
cells that lack Cdc4p activity. The F-box of Cdc4p is a transferable
regulator of protein abundance and when present on a protein
significantly decreases the abundance of that protein. The abundance of
F-box containing proteins is dependent on the abundance of Skp1p. Thus
the F-box has two functions. Firstly, it binds components of the Ub
ligase complex. Secondly it regulates the abundance of Cdc4p. We
speculate that Cdc4p recruitment of the Ub ligase complex stabilizes
Cdc4p. Down regulating Cdc4p activity would be achieved by removing the
Ub ligase complex resulting in a decrease in abundance of Cdc4p.
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