Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT) protein 2

• Published in: PloS one Vol. 5; no. 5; p. e10796
• Main Authors: Gross, David A, Snapp, Erik L, Silver, David L
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.05.2010; Public Library of Science (PLoS)
• Subjects: Adipocytes; Amino Acid Sequence; Amino acids; Analysis; Animals; Biochemistry; Biochemistry/Cell Signaling and Trafficking Structures; Biochemistry/Membrane Proteins and Energy Transduction; Biology; Biosynthesis; Cell Biology; Cell Line; Cholesterol; Conserved Sequence; Cytosol; Diabetes and Endocrinology/Obesity; DNA Mutational Analysis; Droplets; Endoplasmic reticulum; Enzymes; Fluorescent Antibody Technique; Function analysis; Glycosylation; Homology; Humans; Immunofluorescence; Lipids; Localization; Membrane proteins; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Metabolism; Mice; Models, Molecular; Molecular Sequence Data; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation - genetics; Peptide mapping; Physiological aspects; Protein structure; Protein Structure, Tertiary; Proteins; Proteolysis; Regulation; Rodents; Sterols; Storage; Structure-Activity Relationship; Structure-function relationships; Triglycerides; Triglycerides - metabolism; Trypsin; Type 2 diabetes; Zebrafish; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0010796

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.