FIGURE 1.

The ABC half-transporters ALDP and PMP70 bind ACLY and FASN. A, in two independent GST pulldown experiments, recombinant fusion proteins of the C-terminal part of ALDP (GST-C-ALDP, 67 kDa), the C-terminal part of PMP70 (GST-C-PMP70, 62 kDa) or GST alone were incubated with HeLa cell lysates and subjected to Coomassie-stained SDS-PAGE. Prominent bands (arrows) present in the GST-C-ALDP and the GST-C-PMP70 pulldown but not in the control (GST alone) were analyzed by LC/MS. For the identified proteins, the SEQUEST probability values and the SEQUEST protein scores are given (for peptide Xscores see also supplemental Table 1). In GST-C-ALDP pulldown, ACLY (120 kDa) was identified as the binding partner, and in GST-C-PMP70 pulldown FASN (270 kDa) was found. B, co-immunoprecipitation experiments of HeLa cells overexpressing EYFP-ALDP (upper panel) or ECFP-PMP70 (lower panel). Lane 1 shows the co-immunoprecipitation experiments (IP) using a GFP antibody. Immunoblotting (IB) demonstrates that endogenous ACLY co-precipitates with EYFP-ALDP (upper panel) and that endogenous FASN co-precipitates with ECFP-PMP70 (lower panel). Lane 2 shows the control that was incubated with A/G PLUS-agarose but not with antibody. In lane 3, 50 μl of cell lysates as input was analyzed. C, BRET experiments analyzing binary PPI of ALDP, PMP70, ACLY, and FASN. One of eight tag combinations tested that resulted in the highest BRET ratio (supplemental Table 2) is shown. Data are given as the means of n = 2 independent experiments. The dashed line highlights the method-specific threshold for positive PPI of 0.094. Bars representing PPI reported previously in the literature are colored in gray; bars representing novel interactions are highlighted in yellow. D, results of PPI experiments are depicted in a network, with each line connecting two nodes representing an interaction of the proteins reported previously in the literature (gray line), identified by GST pulldown or co-immunoprecipitation and BRET (solid yellow line), or identified by BRET only (dashed yellow line). E, confocal microscopy to examine the co-localization of FASN and peroxisomes. HeLa cells were co-stained with a monoclonal ALDP antibody and anti-mouse-488 secondary antibody to visualize peroxisomes (green) and a polyclonal FASN-antibody and anti-rabbit-Cy3 secondary antibody (red). The yellow color in the overlay image indicates co-localization of FASN and the peroxisome.