Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubi quitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins c opurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subun it of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase- promoting complex. Conditional inactivation of hrt1(ts) results in stabiliz ation of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G(1) /S. Hrt1 assembles into recombinant SCF complexes and individually binds Cd c4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recom binant SCF potently and enables the reconstitution of Cln2 ubiquitination b y recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex acti vate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not app ear to require a reactive thiol. The highly conserved human HRT1 complement s the lethality of hrt1 Delta, and human HRT2 binds CUL-1. We conclude that Cdc5S/Hrt1 comprise a highly conserved module that serves as the functiona l core of a broad variety of heteromeric ubiquitin ligases.