FIGURE 5.

PLIN p.Leu-404fs and p.Val-398fs mutants fail to fully suppress the interaction between ABHD5 and ATGL. The interaction between ABHD5 (Yn-ABHD5) and ATGL (ATGLS47A-Yc) was measured by BiFc in the absence or presence of PLIN1. A, Yn-ABHD5 and ATGL-Yc interact on lipid droplets in the absence of PLIN1, whereas an interaction between them is suppressed by wild-type PLIN1. The PLIN1mutants (p.Leu-404fs and p.Val-398fs) do not suppress interaction between ABHD5 and ATGL as efficiently as wild-type PLIN1. Each panel is a representative image of cells stained with antibodies against Myc (Alexa 594). The top four panels are gray scale intensity profiles, merged in the bottom panel, where blue indicates nuclei (DAPI), red indicates PLIN1, and green indicates the BiFc signal. B, the interaction between ABHD5 and ATGL was quantified as described under “Experimental Procedures.” Data are mean ± S.E. from three independent experiments. Data are normalized in each case to a positive control, i.e. cells expressing YnABHD5 and ATGL S47A-Yc in the absence of exogenous PLIN1. **, p < 0.01. Data shown are YFP fluorescence intensities in the test samples normalized to YFP fluorescence intensities in the positive control, i.e. Yn-ABHD5 and ATGL-Yc cotransfected in the absence of PLIN1. Normalization was done so that samples of equal ABHD5 expression levels were being compared. In the case of wild-type perilipin, the positive control used was 200:200 ng of plasmids encoding Yn-ABHD5:ATGL-Yc, and in the case of the mutants it was 100:100 ng of plasmids encoding Yn-ABHD5:ATGL-Yc. However, normalization was not required to observe a phenotypic difference, i.e. 5-fold greater suppression by wild-type perilipin compared with mutant perilipin. C, representative protein expression of PLIN1, ABHD5, and ATGL in sample sets being compared in the bar graph. _1, a ratio of 100:100 ng; _2, a ratio of 200:200 ng of plasmids encoding YnABHD5:ATGL S47A-Yc.