RanBP2 Modulates Cox11 and Hexokinase I Activities and Haploinsufficiency of RanBP2 Causes Deficits in Glucose Metabolism
Figure 1
The LD of RanBP2 Interacts with Cox11 and HKI
(A) Primary structure of RanBP2 and its structural/functional domains. The N-terminal LD of RanBP2 is underlined.
(B) Sequence alignment of murine and yeast Cox11. The yeast Cox11 C- and N-terminal domains are poorly conserved. Arrow and solid line denote the predicted mitochondrial cleavage site and membrane-spanning domain. The dotted and dashed lines above the aligned sequences represent, respectively, Cox11-N and Cox11-C constructs shown in Figure 1C.
(C) Structure-function analysis of the interaction between the LD of RanBP2 and Cox11. Optimal interaction between the LD and Cox11 occurred in the presence of constructs comprising both the complete LD and Cox11. Although removal of the cytosolic N-terminal (Cox11-C) significantly decreased the interaction with LD, the mitochondrial intermembrane domain of Cox11 (Cox11-C) together with the C-terminal half of LD (LD-C) retained most of the interaction activity. LD-N and LD-C ended and began with the leucine zipper domain of RanBP2. White and black bars denote β-galactosidase activity and growth rates in selective growth medium, respectively. Results shown represent the mean ± SD, n = 3.
(D) GST pull-down assays with the LD of RanBP2 and its leucine zipper domain and retinal extracts. The LD, but not the leucine zipper domain of RanBP2, associate with Cox11 (top panel, lane 1) and HKI (bottom panel, lane 1).
(E) Coimmunoprecipitation of RanBP2 with antibodies against its molecular partners shows that RanBP2 forms a complex in vivo with HKI (lanes 1 and 2), mHsp70 (lane 3), and Cox11 (lane 4). Lanes 5, 6, and 7 are control immunoprecipitation reactions with different antibodies against the RanBP2 domains, KBD, ZnF, and XAFXFG of nucleoporins.
(F) Reciprocal coimmunoprecipitation of HKI with antibodies against RanBP2 (used and shown in (E)).
(G) Reconstitution pull-down assays with purified LD and increasing concentrations of native (top panel), denatured (middle panel), and partially denatured (bottom panel) Cox11, respectively, in the absence and presence of denaturating agent, GnHCl and chaotropic agent, urea. Folding intermediates (lower panel) of Cox11 exhibit the highest binding activity toward the LD of RanBP2.
(H) Similar experiments as in (G) but in the presence of native Cox11 expressed in the absence (top panel) or presence (bottom panel) of CuSO4. The mature isoform of the metallochaperone has an increased affinity toward the LD of RanBP2. LD, leucine-rich domain; LZ, leucine zipper domain; RBD1–4, Ran-binding domains 1–4; ZnF, zinc finger cluster domain; KBD, kinesin (KIF5B/KIF5C)-binding domain; CLD, cyclophilin-like domain; IR, internal repeat domain; CY, cyclophilin domain.