Ubiquitin-carrier protein

From a chemical point of view, the El/ubiquitin thiol ester should be competent to donate ubiquitin to a substrate amino group. In fact, aminoacyl-errzyme thiol esters are used in exactly this way in non-ribosomal polypeptide synthesis, a process that was discovered around the same time as ubiquitin-protein conjugation [5]. In spite of the attractive simplicity of this model, however, biochemical reconstitution studies showed that besides El two additional fractions were required to conjugate ubiquitin to a model substrate. They were called ubiquitin carrier protein (E2) and ubiquitin-protein ligase (E3), respectively, since the respective factors seemed to act sequentially [6]. Interestingly, the E2 factor apparently formed a thiol ester with ubiquitin. Based on these results, Hershko and co-workers proposed the ubiquitin conjugation cascade (Figure 5.1). 

PiCKART, C. M. and Rose, I. A. Eunctional heterogeneity of ubiquitin carrier proteins. J. Biol. Chem. 1985, 260, 1573-81. 

A., Carrano, a., Takizawa, C., Yamanaka, K., Pagano, M., Iwai, K., and CiECHANOVER, A. Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IxrBa. 

Aristarkhov, A., Eytan, E., Moghe, A., Admon, a., Hershko, A., and Ruderman, j. V. E2-C a cydin-selective ubiquitin carrier protein required for the destruction of mitotic cydins. Proc. Natl. Acad. Sd. USA 1996, 93, 4294-99. 

Sullivan, M. L. and Vierstra, R. D. Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis. J. Biol. Chem. 1991, 266, 23878-85. 

These enzymes are also called ubiquitin-carrier proteins or UBCs. Originally, these enzymes were believed only to carry the activated ubiquitin and pass it onto the E3s. Recent studies, however, suggest that at least some E2s can directly conjugate ubiquitin to substrates. ... 

Orian, A., Whiteside, S., Israel, A., Stancovski, I., Schwartz, A.L., and Ciechanover, A. (1995). Ubiquitin-mediated processing of the NF-kB transcriptional activator precursor pl05 Reconstitution of a cell-free system and identification of the ubiquitin-carrier protein, E2, and a novel ubiquitin-protein ligase, E3, involved in conjugation. J. Biol. Chem. 270,21707-21714. 

In a transacylation reaction the ubiquitin moiety is transferred from El-Ub to the SH-group of the ubiquitin-carrier protein E2 to form E2-Ub. 

Genschik, P, A. Durr, and J. Eleck Differential expression of several E2-type ubiquitin carrier protein genes at different developmental stages in Arabidopsis thaliana and Nicotiana sylvestris Molecular Gen. Genetics 244 (1994) 548-556. 

Fig. 5.4 The ubiquitin pathway for protein degradation (Hershko Ciechanover, 1992). Ubiq, ubiquitin E, ubiquiHn-activating enzyme Ej, ubiquitin-carrier protein Ej, ubiquitin-protein ligase CF-1 and CF-2, conjugate-degrading Actors.

Fig. 9.1. Schematic representation of the ubiquitin-proteasome pathway. Ubiquitin moiecuies are activated by an El enzyme (shown green at 1 /3 scaie) in an ATP-dependent reaction, transferred to a cysteine residue (yeiiow) on an E2 or Ub carrier protein and subsequentiy attached to amino groups...

Ubiquitin-dependent protein degradation pathway. ATP hydrolysis is required to attach ubiquitin to the carrier protein El, which then passes the ubiquitin moiety to the activating protein E2. The protein E3 recognizes the NH2-terminal residues and directs E2 to conjugate ubiquitin to lysine residues in the protein. 

NTA - nitrilotriacetic acid CP - carrier protein, b Requires expression as N-terminal fusion with ubiquitin or intein. 

In some families of E3 enzymes, ubiquitin is first transferred from the E2 carrier to an active site cysteine of the E3 enzyme and subsequently to the e-NH2-group of an acceptor lysine on the substrate protein. 

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