nucleotide exchange factor -activation of Arf proteins, which are required for COPI protein binding to Golgi membranes, also inhibited delivery of ATGL to LDs. In addition, recent studies found that ATGL binds to GBF1, a BFA sensitive GEF for Arf1, and that knockdown of COPI components increased LD formation . However, others found that RNAi knockdown of GBF1 had no effect on ATGL association with LDs, which is inconsistent with the BFA results. To explore this issue further, and to determine if other PNPLA family members might utilize the COPI-mediated delivery mechanism, cells expressing ATGL, PNPLA3, or PNPLA5 were treated with BFA before or after induction of LD formation. We confirmed that 
BFA treatment prevented endogenous ATGL from being recruited to LDs, but only when cells were grown in lipoprotein deficient serum for 72 h prior to treatment. Interestingly, BFA did not prevent overexpressed GFP-ATGL or other family members from associating with LDs. Moreover, BFA did not have any influence on the association of PNPLA members with LDs when cells were grown in lipoprotein containing serum prior to treatment. PNPLA Targeting to Lipid Droplets Discussion We found that the C-terminal domains of ATGL, PNPLA3, and PNPLA5 contain two different LTMs. One is a chargedependent basic patch in human PNPLA5 and Drosophila Brummer Lipase. The other encompasses a hydrophobic patch in human ATGL. These results indicate that PNPLA family members interact with, or are recruited to, LDs by diverse mechanisms. Truncation/mutational analysis of PNPLA5 and Drosophila Brummer Lipase identified a charge dependent LTM in the C- PNPLA Targeting to Lipid Droplets 9 PNPLA Targeting to Lipid Droplets length ATGL ML 176 supplier lacking the hydrophobic region in HeLa cells resulted in a decreased LD surface signal and an increased cytoplasmic signal; GFP-tagged N-terminal truncations lacking residues 1319 or a C-terminal truncation lacking residues 361504 were able to localize to LDs at wildtype levels; an N-terminal truncation lacking residues 1360 did not localize to LDs at all. Expressing either full-length ATGL lacking the hydrophobic residues 320360 or a C-terminal truncation lacking residues 320504 resulted in reduced LD localization. Data are plotted as means 6 SEM; $3 experiments/condition, $300 cells counted/experiment. indicates p,0.0001. Fluorescence intensity line plot profiles from line plots in C. Quantitation of LD surface:cytoplasm intensity ratio from line intensity plots for all ATGL constructs. HeLa cells were treated 11325787 as in `A’ except transfections were 18418891 followed by cell fractionation. Wildtype ATGL, but not ATGL lacking the hydrophobic residues 320360, was enriched on LDs as shown by western blotting of isolated LD fractions in comparison to pooled cytoplasmic fractions; quantitation in. Data are plotted as means 6 SEM, n = 3. doi:10.1371/journal.pone.0064950.g006 terminal third of the protein, where each arginine and lysine residue contributes to LD binding. When viewed as a helical wheel, these basic patches may be part of an amphipathic helix. Similar amphipathic helices are known to aid proteins in binding to negatively charged membrane surfaces, including TIP47, which is a LD-associated perilipin family member. Among the PNPLA family members, we observed subtle differences in the LD localization of these lipases. Wild type PNPLA5 localized to LDs in,35% of all cells and thus differs from the other LD localized family members since it targets LDs in a
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