A) Schematic of domain organization of RanBP9 and Flag-tagged deletion constructs: Δ1/N60 (residues 1–392), Δ2, (residues 408–729), and Δ3 (residues 1–107). B) HEK293T cells were transiently transfected with vector control (VC), Flag-tagged RanBP9, Δ1, Δ2, or Δ3 deletion mutants. Equal protein amounts from lysates were immunoblotted for Flag (M2). Note that transient transfection of Flag-RanBP9 yields an N-terminal 60-kDa fragment (Flag-N60), which comigrates with Flag-RanBP9-Δ1/N60.

Both RanBP9 and Δ1/N60 are capable of forming self-interacting complexes. A) Neuro-2A cells were transiently cotransfected with a constant amount of GFP-RanBP9 and increasing concentrations of Flag-RanBP9. After 48 h, lysates were immunoprecipitated with GFP antibody and then immunoblotted with anti-Flag (M2) antibody. B) In similar experiments as above, lysates were immunoprecipitated with anti-Flag M2 antibody and immunoblotted with anti-GFP antibody. Asterisk indicates IgG band. Note the concentration-dependent increase in the interactions between GFP-RanBP9 and Flag-RanBP9. C) Neuro-2A cells were cotransfected with GFP-RanBP9 along with Flag-RanBP9-FL, Flag-Δ1/N60, or empty vector. After immunoprecipitation with anti-Flag M2 antibody, immunoblot detection with GFP antibody showed that Flag-Δ1/N60 interacts with GFP-RanBP9 to a similar extent as full-length Flag-RanBP9.

RanBP9-N60 is robustly increased in AD brains. A) Equal amounts of protein samples from AD brains (n=8) and age-matched normal controls (NC; n=10) were immunoblotted for RanBP9 using anti-RanBP9 antibody. Tubulin was detected with anti-tubulin as a loading control. A representative blot is shown. B) Relative level of full-length RanBP9 (RanBP9-FL) normalized to tubulin. There was no difference in the level of RanBP9-FL normalized to tubulin between AD brain and normal controls. C) Quantitative measurement of the 60-kDa fragment (RanBP9-N60) normalized to tubulin was ∼6-fold higher in AD brains than NC brains, reaching statistical significance. D) When signals from both RanBP9-FL and N60 were combined and normalized to tubulin, there remained ∼75% higher levels in AD brains than in NC brains, which is also highly significant. *P < 0.02.

RanBP9 is proteolytically cleaved to generate RanBP9-N60 in CHO cells. A) CHO-APP751 parental cells and CHO-APP751-flag-RanBP9 stable cells were plated in duplicates, and confluent cultures were subjected to cell lysis and immunoblotting (equal protein amounts). Anti-Flag M2 antibody detected Flag-RanBP9 from stable cells but not from CHO-APP751 control cells (top panel, bottom band), demonstrating that stable cells express transfected RanBP9. In addition to the 90-kDa band expected of full-length RanBP9, an apparent proteolytic N-terminal 60-kDa fragment (RanBP9-N60) was also strongly detected with M2 antibody (top panel, bottom band). When lysates were probed with anti-RanBP9 antibody, endogenous hamster RanBP9 could be detected together with slightly slower migrating transfected human Flag-RanBP9 (bottom panel, arrow). N-terminal 60-kDa fragment was also detected with RanBP9 antibody, which was preferentially increased in RanBP9-transfected cells (bottom panel, bottom band). B) Regulation of RanBP9-N60 formation by cell density and microtubule dynamics. CHO-APP751 cells were plated at indicated densities and lysed in equal volumes, and equal volumes of lysates were probed with anti-RanBP9 monoclonal antibody. Note that N60 is formed only at low density and its formation is gradually reduced as cell density is increased, despite huge excess in protein in higher-density cultures.

RanBP9-Δ1/N60 is relatively more stable and cytoplasmic than RanBP9-FL. A) CHO-APP751 cells were transiently transfected with either RanBP9-FL or RanBP9-Δ1/N60 mutant. After 24 h, cells were stained with M2 antibody, followed by nuclear counterstaining with propidium iodide (PI). While RanBP9-FL was found in both the nucleus and cytoplasm, a significant proportion of cells showed exclusive localization of RanBP9-FL in the nucleus. B) HEK293FT cells were transiently transfected with either Flag-RanBP9-FL or Flag-RanBP9-Δ1/N60, and nuclear and cytoplasmic extracts were analyzed by immunoblotting with the anti-Flag antibody (M2). C) Quantitative measurement of intensity of bands in the nucleus and cytoplasm by Image J software revealed significantly less Δ1/N60 in the nucleus relative to cytoplasm compared with RanBP9-FL (means±se; n=4). *P < 0.01; Student’s t test. D) CHO-APP751 cells stably expressing Flag-RanBP9 or Flag-RanBP9-Δ1/N60 were treated with cycloheximide (100 μg/ml) for the indicated time points. Immunoblot analysis using anti-Flag antibody (M2) showed that the half-life of RanBP9-FL was <1 h, while that of half-life of Δ1/N60 was >3 h.

RanBP9-Δ1/N60 is significantly more potent than RanBP9-FL in APP processing and Aβ generation. A) HEK293FT cells were transiently cotransfected with APP751 and equal amounts of VC, RanBP9-FL (FL), or RanBP9-Δ1/N60 (Δ1) plasmids, and conditioned media and lysates were harvested 48 h after transfection when cells were fully confluent. For detection of Aβ (1), the conditioned medium was immunoprecipitated with B436 monoclonal antibody and immunoblotted with a mixture of 6E10/82E1 antibody. Conditioned media were directly immunoblotted to detect sAPP total (63G; 7), sAPP-α (6E10; 6), and sAPP-β (18957, IBL; 5). In lysates, indicated antibodies were used to detect APP-FL (CT15; 2), APP-CTF total (CT15; 3), APP-CTF-β (6E10; 4), RanBP9 (M2; 8), and D1/N60 (M2; 8). B) Densitometric quantification of Aβ showed an average of ∼3-fold increase by RanBP9-FL compared with vector control. Increase by RanBP9-Δ1/N60 was ∼5-fold, which was also significantly higher than RanBP9-FL. C) Densitometric quantification of the ratio of sAPP-α relative to sAPP total revealed significant differences between VC and Δ1/N60 and also between FL and Δ1/N60 (P<0.01). D) Densitometric quantification of total CTF levels showed significant differences between VC and FL, VC and Δ1/N60, and FL and Δ1/N60. E) Densitometric quantification of the ratio of CTF-β relative to CTF total demonstrated significant differences between VC and FL and VC and Δ1/N60. Graphs show means ± se; n = 4. *P < 0.05, **P < 0.01, ***P < 0.001; 1-way ANOVA with Student-Newman-Keuls post hoc test.