Abstract
Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.
Highlights
The ability to store triglycerides and other non-polar lipids in cytosolic lipid droplets is a ubiquitous biochemical attribute of eukaryotes
FIT1 and FIT2 comprise a unique family of proteins that do not have homology to known proteins or protein domains found in any species
In order to create a foundation for structure-function analysis of Fat Storage-Inducing Transmembrane (FIT) proteins, we sought to create a topological model of FIT proteins by first examining FIT2 as the archetype of the FIT family of proteins since it is the more ancient and conserved form and is expressed in all mammalian tissues, with the highest levels in white and brown adipose tissue [12]
Summary
The ability to store triglycerides and other non-polar lipids in cytosolic lipid droplets is a ubiquitous biochemical attribute of eukaryotes. Several forward genetic screens have been conducted in model organisms or cells to identify proteins important in lipid droplet metabolism and have surprisingly revealed that more than 1% of genes in eukaryotic genomes are involved in lipid droplet biology [9,10,11]. These genetic screens have identified proteins that are involved in regulating lipid droplet lipolysis, neutral lipid biosynthesis, and various other factors which do not yet have assigned roles in lipid droplet biology. Many of these undefined factors may eventually be characterized as having roles in lipid droplet formation
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