FIGURE 1.

Identification of flotillin-2 as a substrate for DHHC5 in neuronal stem cells. A, schematic illustrating quantitative chemo-proteomic profiling of DHHC5-regulated proteins in neuronal stem cells. Isotopically labeled neuronal stem cells were labeled with 17-ODYA, then lysed, and mixed for click chemistry ligation with biotin-azide. Streptavidin-enriched proteins are digested for high resolution proteomic analysis and quantification. B, membrane lysates from 17-ODYA-labeled cells were reacted with rhodamine-azide for SDS-PAGE separation and in-gel fluorescence analysis. Treatment with 1 m hydroxylamine (NH₂OH) releases 17-ODYA from essentially all labeled proteins. Representative MS1 spectra of flotillin-2 from 17-ODYA-enriched membrane proteomes demonstrate specific depletion of palmitoylated flotillins in DHHC5-GT neuronal stem cells. L, light; H, heavy isotope labeling. C, extracted ion chromatographs for FLOT2. D, acyl-biotin exchange analysis of flotillin-2 in NSCs from DHHC5-WT and DHHC5-GT mice. Methods are detailed under “Experimental Procedures.” Lanes labeled Input consisted of 15 μg of whole cell lysate (NSCs) or 25 μg of total brain extract. One-fifth of the total column eluates were loaded. No proteins eluted in the absence of neutral hydroxylamine (NH₂OH), demonstrating the presence of hydroxylamine-sensitive linkages. Rap2b migration differences in input (lanes 1 and 2) versus column eluate (lanes 3 and 5) shown on this gel reflect buffer composition differences in the samples loaded, because the relative positions varied from experiment to experiment and were usually less than what is shown here. E, acyl-biotin exchange analysis of flotillin-2 using whole brain from DHHC5-WT and DHHC5-GT mice. Wild type and DHHC-GT mice were sacrificed by CO₂ narcosis, and the brains were removed, homogenized in lysis buffer, and subjected to acyl-biotin exchange assay as described above. F, quantitation of acyl-biotin immunoblots. Immunoblots from ABE experiments using WT and DHHC5-GT NSCs and mouse brains were quantified using ImageQuant^TM TL software. The values are expressed as the means ± S.D. of the ratio of DHHC5-GT to WT (n = 3, independent experiments). G, 2-bromopalmitate (2-BP) inhibition of S-acylation of flotillin-2 in NSCs. NSCs were pretreated with 2-bromopalmitate at 37 °C for 11 h and then subjected to the acyl-biotin exchange assay. Aliquots (15 μg) of cell lysate (Input) and one-third of total column eluates were subjected to SDS-PAGE and immunoblotting. The results shown were from one of two independent experiments yielding similar results.