Lipolysis is an important metabolic pathway controlling energy homeostasis 
through degradation of triglycerides stored in lipid droplets and release of 
fatty acids. Lipid droplets of mammalian cells are coated with one or more 
members of the PAT protein family, which serve important functions in regulating 
lipolysis. In this study, we investigate the mechanisms by which PAT family 
members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, 
control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in 
adipocytes and several non-adipose cells. We applied fluorescence microscopic 
tools to analyze proteins in situ in cultured Chinese hamster ovary cells using 
fluorescence recovery after photobleaching and anisotropy Forster resonance 
energy transfer. Fluorescence recovery after photobleaching data show that ADFP 
and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas 
perilipin A does not. Differences in protein mobility do not correlate with PAT 
protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. 
Forster resonance energy transfer and co-immunoprecipitation experiments reveal 
that each of the three PAT proteins bind HSL through interaction of the lipase 
with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP 
and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine 
residues within three amino-terminal protein kinase A consensus sequences of 
perilipin A is required for HSL binding and maximal lipolysis. Finally, protein 
kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing 
ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major 
control over HSL-mediated lipolysis when perilipin is the main lipid droplet 
protein.