Structure-function analysis of Fat storage-Inducing Transmembrane (FIT) proteins in mediating lipid droplet formation

Abstract

The process of lipid droplet (LD) formation is an evolutionarily conserved process among all eukaryotes and plays an important role in both cellular physiology and disease. Recently, F&barbelow;at storage-I&barbelow;nducing T&barbelow;ransm&barbelow;embrane proteins 1 & 2 (FITM1/FIT1 and FITM2/FIT2) were discovered as an evolutionarily-conserved family of proteins involved in fat storage. These proteins have distinct tissue distributions, are both exclusively localized to the endoplasmic reticulum, and mediate triglyceride (TAG)-rich LD accumulation when overexpressed. Notably, FIT proteins do not synthesize TAG. Because FIT proteins comprise a novel family of proteins which have no homology to known proteins or protein domains, an approach was utilized to determine structure-function relationships of FIT2. In this thesis, it is demonstrated that FIT1 and FIT2 have six transmembrane domains, and a gain-of-function mutant of FIT2, FLL(157-9)AAA is identified within a domain containing the "FIT signature sequence." FIT2-FLL(157-9)AAA forms significantly larger and more numerous LDs than wild-type FIT2. This phenotype is associated with a conformational change that results in increased solvent accessibility in a cytosolic loop containing amino acid Lys86, suggesting that FIT2 conformation may be modulated by ER lipids or other ER proteins. Here, the hypothesis was tested that FIT proteins bind to TAG to mediate LD formation. Indeed, FIT proteins purified in detergent micelles directly bind to TAG with specificity and saturation binding kinetics. Purified FIT2-FLL(157-9)AAA showed a significant increase in TAG binding. Conversely, FIT1 and a FIT2 partial loss-of-function mutant, N80A, displayed abrogated TAG binding, producing smaller LDs relative to wild-type FIT2. A saturating scanning-alanine mutagenesis screen of FIT2 identified no mutants that retained LD formation capacity in the absence of TAG binding, implying that TAG-binding is required for FIT2 function. In summary, FIT proteins are the first transmembrane domain-containing proteins shown to bind TAG, a function that is important for FIT-mediated LD formation.