Direct binding of triglyceride to fat storage-inducing transmembrane proteins 1 and 2 is important for 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, fat storageinducing transmembrane proteins 1 and 2 (FIT1/FITM1 and FIT2/ FITM2) were discovered as an evolutionarily conserved family of proteins involved in fat storage. In mammals, FIT1 is expressed primarily in skeletal muscle and FIT2 is expressed primarily in adipose, raising the possibility that FIT1 and FIT2 have unique functions. These proteins are exclusively localized to the endoplasmic reticulum (ER) and mediate triglyceride-rich LD accumulation when overexpressed in cells, mouse liver, or muscle. Unlike the ER-resident diacylglycerol O-acyltransferase family of triglyceride-synthesizing enzymes, FITs do not synthesize triglyceride, but rather partition triglyceride into LDs. The mechanism by which FIT proteins mediate this process has not been determined. A simple hypothesis was tested that FIT proteins bind to triglyceride to mediate LD formation. Here, it is shown that FIT proteins purified in detergent micelles directly bind triolein with specificity and saturation-binding kinetics. A FIT2 gain-of-function mutant that formed larger LDs, FLL(157-9)AAA, showed increased binding to triolein relative to wild-type FIT2, whereas FIT1 and a FIT2 partial loss-of-function mutant, N80A, had significantly lower triolein binding and produced smaller LDs. In summary, FIT proteins are transmembrane domain-containing proteins shown to bind triglyceride. These findings indicate that FITs have a unique biochemical mechanism in mediating LD formation and implicates triglyceride binding as important for FIT-mediated LD formation.ipid droplets are cytosolic structures found in cells of all eukaryotes and are composed of a monolayer of phospholipids surrounding a core of uncharged lipids such as triglyceride (TAG) and sterol esters. These structures are considered organelles largely based on the findings that they contain a unique proteome and are dynamic in nature (1). Lipid droplet (LD) formation involves the partitioning of neutral lipids from their site of synthesis at the endoplasmic reticulum (ER) to the cytosol and is considered to be a rapid process (2). The proteins that mediate the partitioning of triglyceride into LDs have not been previously identified; thus, the mechanism of LD formation is not known. Several forward genetic screens were recently conducted in model organisms and cells to identify proteins important in LD biology. These screens have surprisingly revealed that more than 1% of the genes in eukaryotic genomes are involved in LD biology (3-5), underscoring the importance of the LD in normal cellular physiology. Several members of the perilipin family, namely plin2, plin3, and plin4, have been implicated as having roles in LD formation (6-8). However, most evidence supports a role of the plin family in the regulated lipolysis of LDs (9). Interestingly, the ER-resident membrane protein seipin, which is responsible f...

Overexpression of full-length cholesteryl ester transfer protein in SW872 cells reduces lipid accumulation

“…The relative contribution of these two processes to lipid droplet growth is unknown, but there are instances where lipid transfer predominates ( 41,42,47 ). Multiple proteins appear to be involved in the recruitment of TG into lipid droplets in adipocytes, such as fat storage-inducing transmembrane 2 and fat specifi c protein 27 ( 49,50 ). We propose in CETP-expressing species that CETP also participates in this process, and our previous studies in CETP-defi cient cells support this hypothesis ( 14 ).…”

Mature lipid droplets are accessible to ER luminal proteins

“…It is interesting to note, however, that lipid droplets have recently been shown to bud into the ER lumen in cells lacking the fatstorage-inducing transmembrane (FIT) proteins (Choudhary et al, 2015). FIT proteins constitute an evolutionary conserved class of ER proteins that bind triacylglycerol in vitro and they are important for neutral lipid storage in vivo (Gross et al, 2011;Kadereit et al, 2008). Deficiency of FIT2 in the adipose tissue of mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction (Miranda et al, 2014).…”

Keeping FIT, storing fat: Lipid droplet biogenesis

“…It has been found that FITs bind triacylglycerides (TAGs) and diacylglycerol, a precursor of TAGs 23 and thus the FITs may play a role in lipid metabolism that somehow affects LD production. Indeed one of the 2 FIT proteins in yeast has been genetically implicated in lipid metabolism but its role is not known.…”