MAP6d1 is palmitoylated on the N-terminal domain.

<p><i>A</i>, Schematic representation of MAP6d1 constructs. <i>B,</i> NIH/3T3 cells overexpressing MAP6d1 (MAP6d1-CCC) or a MAP6d1 mutant (MAP6d1-GGG) tagged with myc were labeled with [³H]-palmitate. The cells were treated with 100 µM 2-bromopalmitate (Bromo-Pal) or vehicle for 3 h. After immunoprecipitation with anti-myc antibody, proteins were separated by SDS-PAGE and subjected either to autoradiography ([³H]-palmitate) or to immunoblotting (MAP6d1 mAb SLF10). <i>C,</i> Palmitoyl acyltransferase (DHHC) activities on MAP6d1. Each plasmid encoding a DHHC was transfected with the MAP6d1-myc plasmid into HEK-293 cells. After metabolic labeling with [³H]-palmitate, proteins were separated by SDS-PAGE followed by autoradiography (<i>upper panel</i>) and immunoblotting, using an anti-myc antibody to detect MAP6d1 (<i>lower panel</i>). DHHC proteins that enhance MAP6d1 palmitoylation are marked with an asterisk. <i>D,</i>MAP6d1 is palmitoylated on Cys 5, 10 and 11 residues. NIH/3T3 cells were transfected with a plasmid encoding MAP6d1-myc (MAP6d1-CCC) or mutant forms in which one, two or three cysteines were replaced with glycine: MAP6d1-GCC-myc, MAP6d1-CGC-Myc, MAP6d1-CCG-Myc, MAP6d1-GGC-Myc, MAP6d1-GCG-Myc, MAP6d1-CGG-Myc, and MAP6d1-GGG-Myc. Cell extracts were processed as in B. Mutating all 3 MAP6d1 cysteines (MAP6d1-GGG) abolished palmitoylation. <i>E</i>, MAP6d1 protein palmitoylation <i>in vivo:</i> cultured mouse hippocampal neurons (28 days of DIV) were labeled with [³H]-palmitate. MAP6d1 proteins were immunoprecipitated from total cell lysates with MAP6d1 antibody (mAb SLF10). Immunoprecipitated proteins were separated by SDS-PAGE and subjected to autoradiography ([³H]-palmitate) <i>IB</i>: immunoblot, <i>IP</i>: immunoprecipitation, <i>Bromo-Pal</i>: 2-bromopalmitate.