Unlike full-length PIKfyve, the engineered PIKfyve ^CC does not associate with the canonical Vac14/Fig4 complex. (A and B) Rerouting experiments testing the association of the full-length PIKfyve enzyme (A) or the PIKfyve^CC fragment (B) with complex-forming molecular partners, Vac14 and Fig4, using confocal imaging. (A) Recruitable version of the full-length PIKfyve enzyme (iRFP-FKBP-PIKfyve^FL) was co-transfected with mCherry-Vac14 and Fig4-mNG together with an OMM-targeted FRB recruiter (AKAP-FRB-CFP). Rapamycin-induced translocation of FKBP-PIKfyve^FL to the mitochondria also caused translocation of Vac14 and Fig4, which is consistent with their association. (B) Similar experiment showing the inability of the mRFP-FKBP-PIKfyve^CC fragment to co-recruit Vac14 and Fig4 to the OMM. (C and D) shows quantification of the mitochondria-associated signal for the individual constructs from A and B, respectively. These results were reproduced four times. For details of quantification, see Materials and methods (scale bars = 10 µm).