Figure 1.
Identification of Nd1-L as a new interaction partner of KRIT1.
A. Small scale yeast two-hybrid analysis of the interaction of KRIT1 with the C17 clone. Yeast strain AH109 was cotransformed with GAL4DNA-BD and GAL4AD fusion constructs as indicated (left). Cotransformed AH109 cells were streaked out on plates lacking tryptophan and leucine (SD/-Trp/-Leu) or plates lacking tryptophan, leucine, histidine and adenine (SD/-Trp/-Leu/-His/-Ade). The known interaction between p53 protein and SV40 large T-antigen was used as positive control. Empty vectors encoding the GAL4DNA-BD and AD were used as negative control and to exclude aspecific interactions of both K272NT and C17. B. Amino acid sequence of Nd1-L (NP_001034601.1). The sequence of C17 corresponding to Nd1-L (496–642) is underlined. C. Domain structure of the Nd1-L protein. The fragment identified as K272NT interactor in the yeast two-hybrid assay, Nd1-L (496–642), contains a portion of the kelch domain extending from the C-terminal portion of the third to the sixth kelch repeat motif.
Figure 2.
The Nd1-L protein interacts with KRIT1A in vitro.
A. HEK293 cells were transiently transfected with pEGFP vector containing K272NT (left panel) or full-length KRIT1A (right panel). The pull-down assay was performed by incubating transfected (GFP-K272NT, GFP-KRIT1A) or mock-transfected (CTRL) cell lysates with GST-Nd1-L (496–642) immobilized on glutathione-sepharose resin, as indicated. Incubation of transfected cell lysates with GST-glutathione-sepharose was used as negative control. The different incubation mixtures were then analyzed by SDS-PAGE and western blotting with anti-GFP together with aliquots of transfected and mock-transfected cell lysates as controls. B. HEK293 cells were transiently transfected with 2FLAG-Nd1-L. Lysates from transfected (FLAG-Nd1-L) or mock-transfected (CTRL) cells were then incubated with GST-KRIT1A-glutathione-sepharose or with GST-glutathione-sepharose, as indicated. The different incubation mixtures and aliquots of transfected and mock-transfected cells were analyzed by SDS-PAGE and western blotting with anti-FLAG antibodies.
Figure 3.
Nd1-L and KRIT1 associate in HUVEC cells.
Lysates from HUVEC cells were immunoprecipitated with anti-KRIT1 polyclonal antibody or an unrelated antibody as negative control followed by SDS-PAGE and western blotting with an anti-Nd1 antibody (upper panel). Immunoreactive bands at 50 kDa indicate IgG-heavy chains. The IP of KRIT1 was verified by blotting against KRIT1 (lower panel). An aliquot of the HUVEC cells lysate was loaded in the same gel as positive control for the immunodetection phase and to evaluate the relative expression levels of KRIT1 and Nd1.
Figure 4.
The integrity of the KRIT1 FERM domain is required for the interaction with Nd1-L. A.
KRIT1A but not KRIT1B is able to interact with Nd1-L. HEK293 cells were co-transfected with pCR-2FLAG-Nd1-L and pEGFP-KRIT1A or pEGFP-KRIT1B constructs as indicated. 36 hours after co-transfections the cells were harvested and lysed as detailed in Materials and Methods. The co-IP was performed by incubation with anti-KRIT1 or an unrelated antibody as negative control, followed by SDS-PAGE analysis and western blotting with anti-FLAG (upper panel). Mock-transfected cells were used in the assay as controls (CTRL). The IP of EGFP-KRIT1A or EGFP-KRIT1B was verified by anti-GFP western blot (lower panel). Lysates from both transfected and mock-transfected HEK293 cells were loaded in the same gels as positive and negative controls for the immunodetection phase. B. Two different regions of KRIT1 are involved in the interaction with Nd1-L. Full-length KRIT1A, K1AFERM, K1BFERM and K272NT were expressed as GST-fusion proteins and ligated to glutathione-sepharose. Lysates from HEK293 cells overexpressing 2FLAG-Nd1-L were incubated with the different affinity resins as indicated. Incubation with GST-glutathione-sepharose was used as negative control. The different incubation mixtures and an aliquot of the transfected cell lysate were analyzed by SDS-PAGE and anti-FLAG western blotting.
Figure 5.
The interaction with Nd1-L involves two different regions of KRIT1.
A. Schematic representation of the KRIT1 isoforms and deletion mutants used in the assay. B. Yeast two-hybrid analysis of the KRIT1-Nd1-L interaction. pGADT7-Nd1-L was used to co-transform AH109 yeast strain along with different fragments of KRIT1A and KRIT1B isoforms cloned in pGBKT7, as summarized in the associated table (C). Co-transformants were selected on minimal medium lacking Trp and Leu. Protein-protein interaction was assayed on the basis of the ability of Trp+Leu+ transformants to grow in minimal medium lacking His and Ade (activation of HIS3 and ADE2 reporter genes). Yeast cells co-transformed with the pGBKT7 and pGADT7 empty vectors were used as negative control. The interaction between SV40 large T antigen (SV40-T) and p53 was used as positive control. The results of the determination of β-galactosidase activity of Trp+Leu+His+ colonies by filter assay are shown in C. ++++ indicates blue staining of colonies detected within 3 h; +++ blue staining after 4 h; ++ blue staining after 4,5 h; + blue staining after 5,5 h; – no blue staining after 12 h. The results are representative of three independent experiments.
Figure 6.
Nd1-L influences the subcellular localization of KRIT1.
Confocal microscopy analysis of COS-7 (A–C) and A7r5 (D–F) cells transiently transfected with EGFP-KRIT1A (A,D) and Flag-Nd1 (B,E), or both (C,F) cDNA constructs. Nuclei and Flag-Nd1 were visualized with the Hoechst dye and anti-Flag antibody coupled to Alexa Fluor® 633 secondary antibody, respectively. Notice that GFP-KRIT1A is mainly localized into the nucleus when expressed alone (A,D), while it accumulates into the cytoplasm upon co-expression with Flag-Nd1 (C,F). Within the cytoplasm, a significant co-localization of the GFP-KRIT1A and Flag-Nd1-L proteins is also evident. Scale bar represents 15 μm.
Figure 7.
Nd1-L may cooperate with KRIT1 in modulating the expression levels of SOD2.
KRIT1-null (Krit1-) and KRIT1-expressing (Krit1+) MEF cells grown under standard conditions were either mock transfected (−) or transfected with Flag-Nd1-L cDNA (+), and cell lysates were analyzed by Western blot. The expression levels of Flag-Nd1-L were assessed with an antibody against the Flag tag, whereas SOD2 and KRIT1 were detected with specific monoclonal and polyclonal antibodies, respectively. Tubulin served as loading control. Notice that the expression of Flag-Nd1-L induces an upregulation of SOD2 protein levels in KRIT1-expressing but not KRIT1-null MEF cells.