The discovery of perilipin opened a window into the control of cellular lipid metabolism that many investigators in many fields have now climbed through. These studies have established the PAT family as coat proteins for lipid droplets that regulate access to the lipid ester core. A theme that runs throughout studies of the PATs is that they protect the stored lipid esters from uncontrolled hydrolysis by cytoplasmic lipases. Each of the 5 mammalian PAT proteins likely is specialized for specific functions. Perilipin, alone among the mammalian PATs, is known to change its role upon phosphorylation by PKA. In the basal state, perilipin inhibits hydrolysis of triacylglycerol in adipocytes. But when perilipin is phosphorylated on specific serines, it promotes lipolysis. In fact, ectopic expression of perilipin A in CHO cells is sufficient to make lipolysis in those cells responsive to PKA activation[54
]. Even though neither LSD-1 nor LSD-2 in Drosophila are direct orthologs of perilipin, their functions in lipolysis and lipid droplet trafficking, respectively, are similarly modulated by phosphorylation.
The PAT family was originally identified through homology to a highly conserved 100 amino acid domain in the amino terminus of perilipin, the “PAT domain” (). Although all family members can bind to lipid droplets, this droplet targeting is not dependent on the PAT domain. Indeed, there does not appear to be a single domain or motif common to all family members that is responsible for targeting to the droplet surface. Despite its conservation from fungi to mammals, the function of the PAT domain remains unknown. In perilipin A, the PAT domain contains one of the phosphorylation sites thought to be necessary for activation of HSL, yet this phosphorylation site is not conserved in other PAT family members. Finally, the PAT domain is entirely lacking in S3-12; this protein earns its spot in the family based on extensive sequence similarities in other parts of the protein.
Over the past few years, the discoveries of protein-protein interactions between perilipin A and the lipases ATGL and HSL, as well as the ATGL activator CGI-58, have provided clues as to how perilipin might organize lipolysis, but the exact mechanisms remain for detailed elucidation. Now very recent work from Granneman and colleagues has demonstrated the importance of at least functional if not direct physical interactions between OXPAT, CGI-58, and ATGL. It is likely that additional novel protein complexes involving PAT proteins that influence cellular lipid metabolism remain for discovery. Though protein complexes of Drosophila PAT proteins and orthologs of CGI-58, ATGL, and HSL have not yet been reported, these proteins have all been localized to lipid droplets, so it is likely that such complexes play roles in insect lipid metabolism.
It must be remembered that perilipin expression is limited to adipocytes, steroidogenic cells, and perhaps to pathological states of lipid accumulation in vessel macrophages and the liver. Thus, the above observations about the control of lipid stores by perilipins do not apply to most cells in the organism. ADRP is the CPAT protein in these other cells, and its expression at the protein level largely reflects the mass of accumulated neutral lipid. While ADRP may act primarily by excluding lipases from lipid droplet association, as has been suggested for ATGL, it is unclear if there are regulated mechanisms to extract ADRP from the droplet surface, thereby targeting it for proteosomal degradation. An ARF1-dependent mechanism as described by the Fujimoto group is promising [144
]. Indeed, two groups have used genome-wide RNAi screens in Drosophila cells to discover an important role for the ARF-1/COPI trafficking system in the regulation of lipolysis [164
]. In an elegant translation of this discovery back to mammalian cells, Beller and colleagues validated the role of COPI-mediated transport for lipolysis in a mouse liver cell line and found that this pathway modulates the PAT protein composition of lipid droplets[165
]. These studies illustrate the great value of model organisms for the discovery of novel mechanisms in the control of intracellular lipid metabolism.
Intracellular lipid droplets have been compared to both aqueous-cored vesicles and to lipoproteins [24
]. It has been hypothesized that some PAT proteins may be involved in the trafficking of neutral lipid from the site of lipid droplet biogenesis to the site of storage in the perilipin-coated droplet of adipocytes. Roles for PAT proteins in intracellular trafficking have indeed been uncovered. Phosphorylation of perilipin is critical for fragmentation and dispersion of lipid droplets during prolonged lipolytic stimulation. ADRP forms protein complexes with ARF1 and with SNARE proteins, and ADRP deficiency promotes lipid droplet aggregation. TIP47 was originally identified as being involved in the retrieval of the mannose-6-phosphate receptor from the endosomal compartment to the Golgi. Fatvg knockdown results in disrupted trafficking of unidentified vesicles. And the phosphorylation state of LSD-2 determines the directionality of lipid droplet transport in Drosophila embryos. Observations like these make it is likely that novel functions for the PAT proteins will involve the movement of lipid within cells, analogous to the role of apolipoproteins in the movement of lipid between cells and tissues.
Data from cells, mice, and humans, as reviewed above, do not give an unequivocal answer to the question of whether more or less ADRP is beneficial. Because ADRP expression appears to reflect the capacity to store neutral lipids, at least in cell-based gain-of-function studies, the answer to the question of whether less is more may depend on whether lipid storage within a specific tissue is beneficial. Within macrophage foam cells, it may be more advantageous to export cholesterol by reverse transport rather than store it as cholesteryl ester in lipid droplets. Conversely, in skeletal muscle it may be more beneficial to sequester fat in droplets rather than to produce lipotoxic fatty acid metabolites. In this last example, it will be interesting to discover whether differential packaging of lipid droplets in skeletal muscle by different PAT proteins (ADRP versus TIP47 versus S3-12 versus OXPAT) explains the paradoxical observation that the muscle of endurance athletes has increased intramyocellular TAG as does that of sedentary humans with type 2 diabetes.
The finding that single nucleotide polymorphisms in the perilipin gene are associated with obesity and diabetes phenotypes strongly supports the relevance of the PAT family proteins to human lipid and carbohydrate metabolism. It remains to be determined whether genetic variations in the other PAT proteins have similar associations. Why the associations are seen in females but not males remains for investigation.
Future efforts to understand the biology of PAT proteins will require integrated approaches. From the perilipin and ADRP knockout mice, we see that alteration of PAT protein expression leads to secondary effects on gene expression or metabolic function that may not be anticipated. Is the phenotype observed due to loss of a function of the targeted protein? This may or may not be the case. As we have seen, less of one PAT protein may lead to more of another PAT protein on lipid droplets. Thus, the observed phenotype alternatively could be due to decreased levels of the targeted PAT protein on lipid droplets or increased levels of another PAT protein on lipid droplets. Another possibility is that sequestration of the non-targeted PAT protein on lipid droplets may lead to loss of its function in its cytoplasmic compartment (for example, loss of TIP47 for endosomal trafficking). Finally, as has been shown for ATGL, the presence or absence of PAT proteins on lipid droplets affects lipid droplet localization of non-PAT proteins, as well. Thus, it will be important to integrate the mouse genetic models with proteomic studies of isolated lipid droplets, as well as with other unbiased approaches like gene expression microarrays and mass spectrometric analysis of lipids and other metabolites.
The high level of similarity between the regulation of lipolysis in mammals and in insects () highlights the importance of studying the PAT proteins in multiple model systems. Similarities between PAT proteins, both mammalian and non-mammalian () suggests that insights gained in insects, fungi, and slime molds can be translated back to mammalian systems, and vice versa. We anticipate that the problems associated with overnutrition in developed countries may be addressed by novel therapeutic strategies identified through study of PAT proteins in mammalian systems and non-mammalian systems. Undernutrition remains a problem in the underdeveloped world. It is possible that the greatest impact on human health will be made through study of lipid droplets as virulence factors in the pathogenic fungi that infect crop plants, especially if the insights developed for mammalian PAT proteins lead to discoveries that target lipid droplets in pathogenic fungi and thus improve crop yields.
Comparisons of the mammalian and non-mammalian PAT proteins1