CHIP is a U-box-dependent E3 ubiquitin ligase - Identification of Hsc70 asa target for ubiquitylation

Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chap erones such as Hsc/Hsp70 are assisted by cofactors that modulate the foldin g machinery in a positive or negative manner. CHIP (carboxyl terminus of Hs c70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. U sing an in vitro ubiquitylation assay with recombinant proteins, we demonst rate that CHIP possesses intrinsic E3 ubiquitin ligase activity an promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiq uitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70 , primarily with short, noncanonical multiubiquitin chains but has no appre ciable effect on steady-state levels or half-life of this protein. This eff ect may have heretofore unanticipated consequences with regard to the chape roning activities of Hsc70 or its ability to deliver substrates to the prot easome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E 3s.