ATP hydrolysis is required for M1 Spastin to mediate LD–peroxisome contact formation via its PXI region. (A) Diagrams of FP-tagged M1 Spastin mutant constructs. Amino acid number and protein domains are indicated as Fig. 1 A. The mutated residues are labeled in red. (B) Localizations of LDs and peroxisomes in HeLa cells overexpressing mApple-M1 SpastinK388R (top) or mApple-M1 SpastinE422Q (bottom). Representative confocal MIP images are shown. (C) Fraction of LD overlapping peroxisome as described in B and in cells overexpressing mApple-M1 Spastin (red dashed line). mApple-M1 SpastinE422Q-expressing cells were further incubated with nocodazole (noc). Means ± SEM are shown (34–50 cells from at least four independent experiments). ***, P < 0.001; n.s., not significant. (D) Diagrams of a synthetic construct. The red dashed line indicates MIT domain deletion. (E) Colocalization of LDs and mApple-M11–92-197–328 in HeLa cells. Representative confocal images are shown. (F) Association between LDs and peroxisomes in HeLa cells overexpressing mApple- or DsRed2-M11–92-197–328. Representative confocal MIP images are shown. (G) Fraction of LD overlapping peroxisome in HeLa cells overexpressing mApple-M11–92, mApple-M11–92-197–328, DsRed2-M11–92, or DsRed2-M11–92-197–328. DsRed2-M11–92-197–328–expressing cells were further incubated with nocodazole. Means ± SEM are shown (20–37 cells from at least three independent experiments). ***, P < 0.001; n.s., not significant. (H) Diagram of FP-tagged PXI construct (top). mApple-PXI showed cytoplasmic distribution in HeLa cells (bottom). A representative confocal image is shown. (I) Peroxisome purification in HeLa cells transfected with mApple-C1 or mApple-PXI. Crude peroxisome fraction was subjected to density gradient ultracentrifugation to obtain enriched peroxisomes (fractions 4 and 5). Purity of peroxisome and protein levels of overexpressed constructs were assessed by Western blotting using antibodies against ABCD1 and RFP, respectively.
