Figure 7
RMA1 and HRD1 are negative regulators of ABCG5/G8 maturation.
(a–d) Steady-state expression of C-G forms of ABCG5 and ABCG8 proteins in HEK293 cells transfected with empty vector or serial amounts of expression vectors that encode Flag-tagged WT or C42S RMA1 (a) and Myc-tagged WT or C329S HRD1 (c) was analyzed by immunoblotting. * indicates non-specific band. Mature C-G forms of ABCG5 and ABCG8 protein bands after transfection with RMA1 (b) and HRD1 (d) constructs were calculated and data are presented as the percentage of the amount detected in empty vectors-transfected control. *p < 0.05, **p < 0.01, ***p < 0.001, versus empty vector-transfected cells; Dunnett's test (n = 3). Gels have been cropped for clarity; the bands were confirmed by the comparison with full-length gel images (Supplementary Figure S2 and S3) and molecular weight. (e) Schematic flow of RMA1- and HRD1-dependent post-translational regulation of ABCG5 and ABCG8 proteins. RMA1 accelerates the degradation of both ABCG5 and ABCG8 proteins and HRD1 mainly accelerates the degradation of ABCG5 protein, in an E3 activity-dependent manner, while HRD1 accelerates ABCG8 degradation by diminishing the STT3B-dependent post-translational N-glycosylation of ABCG8 in an E3 activity-independent manner. Since ABCG5 N-glycosylation is also mediated by post-translational mechanism (mainly at N584 and partially at N591), HRD1 may inhibit ABCG5 N-glycosylation although the possibility seems to be minor based on the data. Hence, both RMA1 and HRD1 are negative regulators of ABCG5/ABCG8 maturation.
