MIB1 colocalizes with RYK on intracellular membranes. (A–D) Single-plane confocal images of immunofluorescent labeling of HEK293T cells transiently transfected with cDNA encoding full-length glue-tagged RYK and flag-tagged MIB1 or variants of MIB1. RYK is primarily localized to the plasma membrane when expressed alone (A). However, when overexpressed with MIB1 (B), RYK and MIB1 colocalize to intracellular membranous organelles that are also positive for Rab5. Catalytically impaired MIB1-C997S colocalizes with RYK in aggregates at or near the plasma-membrane (C). In contrast, overexpression of MIB1 lacking all three enzymatic domains MIB1-ΔRN3 (D) minimally affects RYK surface localization and colocalizes at the sites of cell protrusions. (E) Quantification of immunocytochemistry demonstrating that MIB1 and MIB1-C997S reduce the distribution of RYK along the plasma membrane. Data are mean + SEM (error bars). *, P < 0.005 (Student’s t test, n = 4). Bar, 10 µm.