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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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A conserved tract of amino acids in transmembrane domain 4 of FITs.Alignment of partial sequences of FIT2 and FIT1 from various species showing the “FIT signature sequence” is indicated within the red box. The most conserved group of residues in this domain comprises the FLL sequence, whose hydrophobic character was retained throughout evolution. Based on our experimentally determined topological model of FIT proteins (Figure 1 and 2), the FIT signature sequence is located in transmembrane domain 4.
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pone-0010796-g003: A conserved tract of amino acids in transmembrane domain 4 of FITs.Alignment of partial sequences of FIT2 and FIT1 from various species showing the “FIT signature sequence” is indicated within the red box. The most conserved group of residues in this domain comprises the FLL sequence, whose hydrophobic character was retained throughout evolution. Based on our experimentally determined topological model of FIT proteins (Figure 1 and 2), the FIT signature sequence is located in transmembrane domain 4.

Mentions: Transmembrane domain 4 of FITs contains the most highly conserved residues throughout the FIT protein family which can be considered a FIT signature sequence (Fig. 3). Using FIT2-V5 as an archetype for our studies of FIT function, we performed alanine mutagenesis on this domain. Single or paired conserved residues were mutated to alanine, expressed in HEK293 cells, and lipid droplets were visualized by fluorescence confocal microscopy in order to characterize alterations in phenotype. To visualize lipid droplets we utilized the neutral lipid fluorescent dye BODIPY 493/503, commonly used to visualize and quantify lipid droplets due to its high specificity for lipid droplets [18]. This stain was recently and successfully used in a high-throughput forward genetic screen to identify genes important in lipid droplet biology [10]. In order to determine that cells having lipid droplets expressed FIT2, FIT2 expressing cells were identified by immunofluorescence. Of the mutants generated, FLL(157-9)AAA (abbreviated as FLL) showed a pattern of increased number of lipid droplets and larger lipid droplets compared to wild-type FIT2 when subjected to BODIPY 493/503 staining and confocal fluorescence microscopy (Fig. 4A). The observed lipid droplet phenotype of cells expressing FLL could not be explained by either expression level of FLL relative to wild-type FIT2, its subcellular localization (Fig. 5 lanes 1 & 2, Fig. S3, and Fig. S5), or transfection efficiency (90% for both wild-type and FLL). Mock transfected cells served as a negative control for lipid droplet staining, since HEK293 cells rarely exhibited lipid droplets. DGAT2 overexpression served as a positive control and resulted in an abundance of small lipid droplets. Quantification of lipid droplet number and size clearly indicated that expression of the FLL mutant resulted in a 5-fold increase in mean lipid droplet size (WT, 0.8±0.2 µm3; FLL, 4.0±0.3 µm3; p = 3.68×10−6) (Fig. 4B) and a 5-fold increase in lipid droplet number compared to cells expressing wild-type FIT2 (Fig. 4C). As expected, mock-transfected cells rarely exhibited droplets, while DGAT2-expressing cells had an average of 21 small lipid droplets per cell (average volume, 0.55±0.08 µm3) (Fig. 4B, C). Despite the 5-fold increase in both lipid droplet number and size in cells expressing FLL mutant, triglyceride levels were only increased 1.8-fold compared to cells expressing WT FIT2. Triglyceride levels in cells expressing FLL or WT FIT2 were moderately increased relative to mock-transfected control cells (3-fold and 1.6-fold, respectively) compared to the greater than 10-fold increase in cells expressing DGAT2 (Fig. 4D). This finding is consistent with our previous finding that FIT2 does not mediate triglyceride biosynthesis, a biochemical function mediated by the DGAT enzymes [13], but rather mediates the partitioning of cellular triglycerides into lipid droplets [12]. No significant changes in various phospholipid species were found (Fig. 4D). Taken together, the data indicate that the FLL mutation imparts a gain-of-function on FIT2 resulting in increased lipid droplet accumulation.


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

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

A conserved tract of amino acids in transmembrane domain 4 of FITs.Alignment of partial sequences of FIT2 and FIT1 from various species showing the “FIT signature sequence” is indicated within the red box. The most conserved group of residues in this domain comprises the FLL sequence, whose hydrophobic character was retained throughout evolution. Based on our experimentally determined topological model of FIT proteins (Figure 1 and 2), the FIT signature sequence is located in transmembrane domain 4.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875400&req=5

pone-0010796-g003: A conserved tract of amino acids in transmembrane domain 4 of FITs.Alignment of partial sequences of FIT2 and FIT1 from various species showing the “FIT signature sequence” is indicated within the red box. The most conserved group of residues in this domain comprises the FLL sequence, whose hydrophobic character was retained throughout evolution. Based on our experimentally determined topological model of FIT proteins (Figure 1 and 2), the FIT signature sequence is located in transmembrane domain 4.
Mentions: Transmembrane domain 4 of FITs contains the most highly conserved residues throughout the FIT protein family which can be considered a FIT signature sequence (Fig. 3). Using FIT2-V5 as an archetype for our studies of FIT function, we performed alanine mutagenesis on this domain. Single or paired conserved residues were mutated to alanine, expressed in HEK293 cells, and lipid droplets were visualized by fluorescence confocal microscopy in order to characterize alterations in phenotype. To visualize lipid droplets we utilized the neutral lipid fluorescent dye BODIPY 493/503, commonly used to visualize and quantify lipid droplets due to its high specificity for lipid droplets [18]. This stain was recently and successfully used in a high-throughput forward genetic screen to identify genes important in lipid droplet biology [10]. In order to determine that cells having lipid droplets expressed FIT2, FIT2 expressing cells were identified by immunofluorescence. Of the mutants generated, FLL(157-9)AAA (abbreviated as FLL) showed a pattern of increased number of lipid droplets and larger lipid droplets compared to wild-type FIT2 when subjected to BODIPY 493/503 staining and confocal fluorescence microscopy (Fig. 4A). The observed lipid droplet phenotype of cells expressing FLL could not be explained by either expression level of FLL relative to wild-type FIT2, its subcellular localization (Fig. 5 lanes 1 & 2, Fig. S3, and Fig. S5), or transfection efficiency (90% for both wild-type and FLL). Mock transfected cells served as a negative control for lipid droplet staining, since HEK293 cells rarely exhibited lipid droplets. DGAT2 overexpression served as a positive control and resulted in an abundance of small lipid droplets. Quantification of lipid droplet number and size clearly indicated that expression of the FLL mutant resulted in a 5-fold increase in mean lipid droplet size (WT, 0.8±0.2 µm3; FLL, 4.0±0.3 µm3; p = 3.68×10−6) (Fig. 4B) and a 5-fold increase in lipid droplet number compared to cells expressing wild-type FIT2 (Fig. 4C). As expected, mock-transfected cells rarely exhibited droplets, while DGAT2-expressing cells had an average of 21 small lipid droplets per cell (average volume, 0.55±0.08 µm3) (Fig. 4B, C). Despite the 5-fold increase in both lipid droplet number and size in cells expressing FLL mutant, triglyceride levels were only increased 1.8-fold compared to cells expressing WT FIT2. Triglyceride levels in cells expressing FLL or WT FIT2 were moderately increased relative to mock-transfected control cells (3-fold and 1.6-fold, respectively) compared to the greater than 10-fold increase in cells expressing DGAT2 (Fig. 4D). This finding is consistent with our previous finding that FIT2 does not mediate triglyceride biosynthesis, a biochemical function mediated by the DGAT enzymes [13], but rather mediates the partitioning of cellular triglycerides into lipid droplets [12]. No significant changes in various phospholipid species were found (Fig. 4D). Taken together, the data indicate that the FLL mutation imparts a gain-of-function on FIT2 resulting in increased lipid droplet accumulation.

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