The Amino and Carboxyl Termini of Perilipin A Facilitate the Storage of Triacylglycerols

Abstract

Perilipin A is the most abundant lipid droplet-associated protein in adipocytes and serves important functions in regulating triacylglycerol levels by reducing rates of basal lipolysis and facilitating hormonally stimulated lipolysis. We have previously shown that the central region of perilipin A targets and anchors it to lipid droplets, at least in part via three moderately hydrophobic sequences that embed the protein into the hydrophobic core of the droplet. The current study examines the roles of the amino and carboxyl termini of perilipin A in facilitating triacylglycerol storage. Amino-and carboxyl-terminal truncation mutations of mouse perilipin A were stably expressed in 3T3-L1 preadipocytes, which lack perilipins. Triacylglycerol content of the cells was quantified as a measure of perilipin function and was compared with that of cells expressing full-length perilipin A or control cells lacking perilipins. The aminoterminal sequence between amino acids 122 and 222, including four 10 -11-amino acid sequences predicted to form amphipathic ␤-strands and a consensus site for cAMP-dependent protein kinase, and the carboxyl terminus of 112 amino acids that is unique to perilipin A were critical to facilitate triacylglycerol storage. The precocious expression of full-length perilipin A in 3T3-L1 preadipocytes aided more rapid storage of triacylglycerol during adipose differentiation. By contrast, the expression of highly truncated amino-or carboxylterminal mutations of perilipin failed to serve a dominant negative function in lowering triacylglycerol storage during adipose differentiation. We conclude that the amino and carboxyl termini are critical to the function of perilipin A in facilitating triacylglycerol storage.Triacylglycerols packaged within the lipid droplets of adipocytes provide the most abundant form of stored energy in the body. Lipid droplets are spherical structures composed of a core of neutral lipids coated by a phospholipid monolayer into which specific proteins are embedded. The study of lipid dropletassociated proteins is an emerging field of inquiry. Only a few of the component proteins have been identified, and little is known about the structural properties that define lipid dropletassociated proteins and facilitate their functions in lipid metabolism. The aim of the current study is to begin to define functional domains of perilipins, the first identified mammalian lipid droplet-associated proteins.Perilipins coating the surfaces of adipocyte lipid droplets serve as gatekeeper proteins controlling both the storage and release of triacylglycerol. The translation of alternatively spliced mRNAs produces three protein isoforms of perilipin: perilipins A, B, and C (1, 2). Perilipin A is the most abundant protein located at the surfaces of lipid droplets in adipocytes and is found at lower relative abundance on lipid droplets in steroidogenic cells of the adrenal cortex, testes, and ovaries (3); perilipin B is found at lower levels in both types of cells, and perilipin C is selectively expre...

Perilipin A Mediates the Reversible Binding of CGI-58 to Lipid Droplets in 3T3-L1 Adipocytes

“…13) lacking N-terminal sequences from amino acids 1-81, perilipin mutation N4 (12) (also called mutation N7; Ref. 13) lacking N-terminal sequences from amino acids 1 to 222, perilipin mutation C1 (12, 13) lacking C-terminal amino acids 490 -517, and perilipin mutation C2 (12, 13) lacking C-terminal amino acids 430 -517.…”

“…Stable Expression of Intact Perilipin A and Mutated Perilipins in 3T3-L1 Preadipocytes-Cells stably expressing intact perilipin A (26), or mutated perilipins lacking portions of the N and C termini were prepared previously (12,13); these cell lines include cells stably expressing perilipin mutation N1 (12) (also called mutation N3; Ref. 13) lacking N-terminal sequences from amino acids 1-81, perilipin mutation N4 (12) (also called mutation N7; Ref.…”

“…Mutated forms of perilipin used in this study were previously shown to target to lipid droplets (12,13). CGI-58 localized to the surfaces of lipid droplets in cells stably expressing intact perilipin A (Fig.…”

“…Studies in perilipin knockout mice have established an important role for perilipins in regulating lipolysis in adipocytes, and hence, controlling the mass of triacylglycerol deposited in adipose tissue (10, 11). The mechanisms by which perilipins maintain control of adipocyte triacylglycerol metabolism are poorly understood, although recent mutagenesis studies have identified structural motifs important to perilipin function in triacylglycerol storage and lipolysis (12)(13)(14)(15)(16)). An attractive model consistent with these data suggests that perilipins restrict the access of cytosolic lipases to triacylglycerol stored within lipid droplets, and thus suppress the rate of basal lipolysis under conditions when triacylglycerol storage is favored.…”

Cloning, chromosome mapping and expression pattern of porcinePLINandM6PRBP1genes

“…The human PLIN gene has two mRNA variants that yield two proteins, perilipin A and B. Perilipin A is the predominant protein isoform and plays key roles in facilitating both the storage and hydrolysis of triacylglycerol (TGA) [7]. Based on the different splicing sites present in the mouse and human genes, we designed different primers to try to amplify porcine PLIN mRNA variants in different tissues, but failed to find other variants.…”