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

Gross DA, Snapp EL, Silver DL - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.

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.

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Model for conformational change in FIT2 FLL mutant.The FIT2 FLL mutant has an altered conformation relative to wild-type FIT2 that results in increased solvent accessibility of residues in cytosolic loop 2 containing residue K86. This conformational change may facilitate increased partitioning of triglyceride into nascent cytosolic lipid droplets or allow for the creation of larger lipid droplets by undetermined mechanisms.
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pone-0010796-g007: Model for conformational change in FIT2 FLL mutant.The FIT2 FLL mutant has an altered conformation relative to wild-type FIT2 that results in increased solvent accessibility of residues in cytosolic loop 2 containing residue K86. This conformational change may facilitate increased partitioning of triglyceride into nascent cytosolic lipid droplets or allow for the creation of larger lipid droplets by undetermined mechanisms.

Mentions: We hypothesized that the FLL mutant has an altered conformation relative to wild-type FIT2. We used limited trypsin digestion to test this hypothesis. To perform this method, which has been used successfully to study lipid-induced changes in two other ER resident membrane proteins, HMG-CoA Reductase and SCAP [19], [20], we needed to develop an antibody to a luminal loop near the FLL sequence. Taking advantage of our topological model of FIT2, we developed antibodies to the second luminal loop of FIT2. Our studies revealed that trypsin digestion of FIT2 and the FLL mutant resulted in a trypsin resistant fragment of ∼15 kDa (designated P2) due to cleavage at K86 and K256/R257. Importantly, the amount of P2 fragment generated by digestion of the FLL mutant was increased relative to wild-type FIT2, indicating an altered conformation of this mutant. We interpret these findings to indicate that the FLL residues in transmembrane domain 4 stabilize a specific conformation that attenuates or regulates FIT2 activity. Substitution of these large non-polar side chains with methyl groups either increases solvent accessibility around K86 and/or permits greater antibody affinity in the second luminal loop of FIT2, as indicated in limited trypsin digestion experiments, correlating with increased FIT2 activity (Fig. 7). It is tempting to speculate that the activity of wild-type FIT2 is regulated by conformational changes induced by ER membrane lipids or interaction with other ER proteins. Such modes of regulation have been clearly demonstrated for Insig, SCAP and HMG-CoA Reductase [22], [23]. Whether FIT2 is similarly regulated will require further study into this recently identified family of proteins.


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

Gross DA, Snapp EL, Silver DL - PLoS ONE (2010)

Model for conformational change in FIT2 FLL mutant.The FIT2 FLL mutant has an altered conformation relative to wild-type FIT2 that results in increased solvent accessibility of residues in cytosolic loop 2 containing residue K86. This conformational change may facilitate increased partitioning of triglyceride into nascent cytosolic lipid droplets or allow for the creation of larger lipid droplets by undetermined mechanisms.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875400&req=5

pone-0010796-g007: Model for conformational change in FIT2 FLL mutant.The FIT2 FLL mutant has an altered conformation relative to wild-type FIT2 that results in increased solvent accessibility of residues in cytosolic loop 2 containing residue K86. This conformational change may facilitate increased partitioning of triglyceride into nascent cytosolic lipid droplets or allow for the creation of larger lipid droplets by undetermined mechanisms.
Mentions: We hypothesized that the FLL mutant has an altered conformation relative to wild-type FIT2. We used limited trypsin digestion to test this hypothesis. To perform this method, which has been used successfully to study lipid-induced changes in two other ER resident membrane proteins, HMG-CoA Reductase and SCAP [19], [20], we needed to develop an antibody to a luminal loop near the FLL sequence. Taking advantage of our topological model of FIT2, we developed antibodies to the second luminal loop of FIT2. Our studies revealed that trypsin digestion of FIT2 and the FLL mutant resulted in a trypsin resistant fragment of ∼15 kDa (designated P2) due to cleavage at K86 and K256/R257. Importantly, the amount of P2 fragment generated by digestion of the FLL mutant was increased relative to wild-type FIT2, indicating an altered conformation of this mutant. We interpret these findings to indicate that the FLL residues in transmembrane domain 4 stabilize a specific conformation that attenuates or regulates FIT2 activity. Substitution of these large non-polar side chains with methyl groups either increases solvent accessibility around K86 and/or permits greater antibody affinity in the second luminal loop of FIT2, as indicated in limited trypsin digestion experiments, correlating with increased FIT2 activity (Fig. 7). It is tempting to speculate that the activity of wild-type FIT2 is regulated by conformational changes induced by ER membrane lipids or interaction with other ER proteins. Such modes of regulation have been clearly demonstrated for Insig, SCAP and HMG-CoA Reductase [22], [23]. Whether FIT2 is similarly regulated will require further study into this recently identified family of proteins.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.

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.

Show MeSH