Biology Reference
In-Depth Information
Abstract
The lipid droplet (LD) is an evolutionarily conserved organelle composed primarily
of triglycerides (TAG) and cholesteryl esters. Recently,
Fa
t storage-
I
nducing
T
rans-
membrane proteins 1 & 2 (FITM1/FIT1 and FITM2/FIT2) were discovered as a con-
served family of proteins involved in fat storage. FIT1 and FIT2 are both localized to
the endoplasmic reticulum, but have distinct tissue distributions. FIT proteins medi-
ate TAG LD accumulation when overexpressed, but do not synthesize TAG. FIT pro-
teins function by partitioning newly synthesized TAG into LDs. In order to
understand the mechanism by which this occurs, a method was developed to purify
FIT proteins from insect cells in detergent micelles. The ability of purified FIT pro-
teins to bind TAG and other neutral lipids was tested in detergent micelles, demon-
strating lipid specificity and saturation binding. These techniques can be applied to a
variety of proteins in lipid biology in an effort to try to reconstitute a mechanism of
action or protein activity. The methods that will be discussed here can also be scaled
to either screen a library of mutant proteins for binding to a particular compound or
utilized to delineate structural requirements of ligands that are important for protein-
ligand interactions. Here, we present a detailed description of the purification pro-
tocol and micellar protein-ligand binding experiments and their possible
applications.
INTRODUCTION AND RATIONALE
Lipid droplets (LDs) are organelles with a unique structure composed of a hydropho-
bic core that is encapsulated by a phospholipid monolayer with a diverse proteome.
The lipid core is composed of relatively hydrophobic, minimally polar lipids includ-
ing triglycerides (TAG), cholesteryl esters (CE), diacylglycerol (DAG), and
monoacylether-DAG (
Wolins, Brasaemle, & Bickel, 2006
). The predominant com-
ponents of LDs in most cell types are TAG and CE. Accumulation of excess TAG in
the form of LDs in adipose, liver, and skeletal muscle is associated with the devel-
opment of insulin resistance, type 2 diabetes, and the metabolic syndrome (
Savage,
Petersen, & Shulman, 2007a, Savage et al., 2007b
).
TAG biosynthesis is a highly regulated process beginning with fatty acid and
glycerol synthesis and is reviewed in detail elsewhere (
Coleman & Lee, 2004
).
TAG can be synthesized from DAG by a variety of enzymes with acyltransferase
activity, including DAG:acyl-CoA acyltransferases 1 and 2 (DGAT1 and DGAT2),
sn-
1,2(2,3)-DAG transacylase, wax ester synthase/DGAT, or lecithin-DAG transa-
cylase (
Coleman & Lee, 2004
). In mammals, the most important isoform of these
enzymes is DGAT2. Deficiency of DGAT2 in mice results in more severe reductions
in TAG levels than DGAT1 deficiency, consistent with the finding that it is more
highly expressed in tissues that make large amounts of TAG (
Cases et al., 2001;
Farese, Cases, & Smith, 2000; Stone et al., 2004
). Interestingly, conventional
