MRB8620 is required for MRB1 core integrity. (A) The effect of MRB8620 and GAP1 depletion on the sedimentation of GAP1, MRB3010, TbRGG2, and RECC subunit KREPA3. Mitochondrial lysates from the parental cell line (top images), RNAi-silenced MRB8620 (middle images), and GAP1 (lower images) T. brucei were loaded on 10%–30% glycerol gradients for their fractionation. Alternate gradient glycerol fractions were analyzed by immunodecoration of Western blots with the indicated antibodies indicated on the right. To the right of the 23rd fraction (loading) is a sample from the lysis step before loading onto the gradient to demonstrate equivalent amounts of protein were used among samples. (B) Scheme of MRB1 core complex components in cells harboring MRB8620 RNAi with MRB3010 V5 tagged at an endogenous allele. MRB3010-V5 and associated proteins were isolated by Protein G-Dynabead from extracts of mitochondria from either uninduced (tet−) or RNAi-induced (tet+) MRB8620 RNAi cells. Both input and elutions (indicated above images) were analyzed by Western blot for MRB1 complex components using the antibodies indicated on the right. (C) As in B, except that the cells harbor the GAP1 RNAi construct with MRB3010 V5 tagged at an endogenous allele.
