Biology Reference
In-Depth Information
FIT protein overexpression studies indicated that FIT proteins induce the accu-
mulation of TAG-rich LDs, without augmenting TAG biosynthetic rates, by medi-
ating the partitioning of TAG into LDs (
Gross et al., 2010; Kadereit et al., 2008
). In
HEK293 cells, FITs neither altered the expression of genes important for fatty acid
oxidation or TAG biosynthesis, nor did they alter lipolysis of TAG found in LDs.
Taken together, these data indicate that FIT proteins play a unique role to partition
newly synthesized TAG into LDs.
Since FIT proteins were found to be involved in LD accumulation, it was hypoth-
esized that they may play a role in adipocyte differentiation. However, no changes in
PPAR-
g
levels or PPAR-
g
targets were observed in 3T3-L1 cells deficient in FIT2.
Importantly, FIT2 knockdown in preadipocytes led to a reduction in total cellular
LDs and TAG levels and reduced TAG biosynthesis throughout a time course of
adipocyte differentiation (
Kadereit et al., 2008
). Thus, the initial identification, clon-
ing, and characterization of FIT1 and FIT2 indicated that these proteins are localized
to the ER, important for TAG storage and LD accumulation, but not involved in TAG
biosynthesis. Their knockdown does not affect adipocyte differentiation; however, it
significantly abrogates the accrual of TAG in these cells.
Since FIT proteins have no homology to proteins of known function or protein
domains, it is impossible to infer a mechanism based on sequence similarity to a fam-
ily of well-characterized proteins. Given the finding that FIT proteins were found to
partition newly synthesized TAG into LDs, we hypothesized that FIT proteins bind
directly and specifically to TAG in order to mediate LD formation at the ER. We
have recently tested this hypothesis using purified FIT1 and FIT2 in detergent
micelles and demonstrated that FIT proteins bind to TAG in order to mediate LD
biogenesis and that this function is required for FIT-mediated LD formation. Puri-
fication of integral membrane proteins and membrane-associated proteins can be
particularly useful for characterizing novel protein players in LD biology.
Purified proteins important in LD biology can be used to study the process of LD
biogenesis with
in vitro
reconstitution assays, determine biochemical or enzymatic
functions of LD-associated proteins, and can be produced in large quantities for
biophysical studies or crystallization trials. This review aims to highlight the
techniques that were used to purify the multispanning ER-localized transmembrane
proteins FIT1 and FIT2 and study their lipid-binding characteristics. We have used
the purification and lipid-binding assay techniques that were developed for FIT
proteins for other transmembrane proteins with success, suggesting that purification
of membrane proteins from insect cells in detergent micelles can be used to study a
variety of other membrane-associated or integral membrane proteins involved in LD
biology. Our studies highlight methods that demonstrate the power of lipid-binding
studies in determining ligand preference, an important step in uncovering the
mechanism by which lipid-binding proteins function. However, there are still
limitations in determining precise kinetic constants and detecting conformational
changes in detergent micelles due to stoichiometric behaviors of the system. In this
review, we also highlight techniques that could be used in an effort to reconstitute LD
formation.
