The LD of RanBP2 Interacts with Cox11 and HKI
The LD of RanBP2 (Figure 1A) is a large and orphan domain of ~700 residues (~80 kDa), for which no molecular partners have been identified until this date. Brain and retina yeast two-hybrid libraries were screened with LD. Cox11 was identified as a partner to this domain (Figure 1B and 1C). Cox11 is a metallochaperone implicated in cytochrome c oxidase assembly [30,31]. Structure-function analysis of the interaction between mCox11, LD of RanBP2, and subdomains thereof, with quantitative yeast two-hybrid assays [32], showed optimal interaction between the intact LD and Cox11 proteins (Figure 1C). Pull-down assays of retinal extracts with glutathione S-transferase (GST)-LD precipitates a sodium dodecyl sulfate-resistant dimer isoform of Cox11 (Figure 1D, top panel), which does not bind to GST-LDZIP alone. In addition to Cox11, we also found that other mitochondrial components such as the outer membrane-associated protein, HKI [33] (Figure 1D, bottom panel) and mHsp70 (unpublished data), associated with LD of RanBP2. This association was highly specific toward the HKI isoform, because HKII, HKIII, and glucokinase did not interact with the LD of RanBP2 (unpublished data). The interaction of Cox11, HKI, and mHsp70 with RanBP2 occurred in vivo in retinal extracts, since antibodies against these and RanBP2 coimmunoprecipitated RanBP2 (Figure 1E) and HKI (Figure 1F), respectively, and these interactions were observed across different tissues (Figure S1). Since RanBP2 exhibits chaperone activity, we assessed whether the interaction between the LD of RanBP2 and Cox11 was direct and the chaperone activity of LD toward folding species of Cox11. Reconstitution binding assays were carried out between purified LD and Cox11, fully and partially denatured with GnHCl and urea, respectively, and native Cox11 (Figure 1G and 1H). Partial denatured Cox11 exhibits significantly higher and concentration-dependent binding affinity toward LD compared with the native and fully denatured Cox11 (Figure 1G). In addition, native Cox11 purified upon expression in the presence of CuSO4 (a prosthetic group tightly bound to Cox11) [31], shows significantly higher binding activity toward the LD of RanBP2, than in the absence of CuSO4 (Figure 1H).
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.