FIGURE 5.

Role of gp78 positively charged patches in UBC7-gp78-mediated CYP3A4 ubiquitination. A, schematic linear representation of the cytosolic gp78 C-terminal 309–643-residue domains, the deletion constructs, and positively charged QKHR patches containing Lys residues either found or suspected to be cross-linked to CYP3A4 and chosen for site-directed mutagenesis (locations marked by a red asterisk). B, influence of the gp78 deletion mutants shown in A or gp78K313A single mutant on in vitro UBC7-gp78-mediated CYP3A4 ubiquitination. Control (CT) incubations were conducted in parallel in the absence of UBC7 (−E2), gp78 (−E3), or CYP3A4 (−3A4) for 90 min. C, influence of the Ala mutation of gp78 N-terminal Arg-307–Arg-308–Arg-310–Arg-311–His-312–Lys-313 patch (313PT) on UBC7-gp78-mediated CYP3A4 ubiquitination over a 0–90-min incubation period. Ubiquitination reactions containing either the wild type gp78C (gp78CWT), gp78-C1 (gp78 deletion construct lacking its G2BR and VIM domains), or its Ala 313PT mutant were carried out, and CYP3A4 ubiquitination was examined at 0, 30, 60, and 90 min as detailed under “Experimental Procedures.” D, relative UBC7-gp78-mediated CYP3A4 ubiquitination over a 90-min incubation period of gp78CWT or its 313PT, R594V/K595A/R596V (595VAV), R594A/K595A/R596A (595AAA), and Q584A/R585A/K586A (586PT) mutants singly or in combination. Control reactions containing no CYP3A4 substrate (−3A4), or excluding the E1, E2, or E3 were also run in parallel. E, effects of the gp78 patch (PT) mutants shown in D on basal UBC7-gp78 ubiquitin chain processing in the absence of added CYP3A4 is shown as evidence for the lack of their effects on basal E2-E3 interactions. Note that unlike the 10–20-s exposures required for the detection of CYP3A4 ubiquitination (A–D), the detection of basal E2-E3 ubiquitin chain processing required 5–10-min exposures.