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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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Membrane topology of murine FIT2 and FIT1.A and C, FIT2 and FIT1 topological models with N- and C-termini oriented toward the cytosol and six transmembrane domains with a large second luminal loop. The amino acid positions of insertion of FNF tags for the determination of topology by glycosylation site mapping and indirect immunofluorescence are indicated. As indicated, all constructs contain C-terminal V5 and polyhistidine epitope tags. B and D, Increased molecular weights of specific FNF-V5 constructs relative to wild-type FIT2 or FIT1 (indicated by asterisks) detected by Western blot analysis indicated potential glycosylation and luminal orientation. Decreased molecular weight of constructs following PGNaseF treatment indicated glycosylation and luminal orientation. Asterisks indicate glycosylated forms of FIT2 and FIT1 constructs. The multiple asterisks indicated for the FIT2 construct 51 is due to two tandem FNF glycosylation sites inserted into that construct (arrow indicates non-glycosylated construct 51). B and D are representative of two independent experiments.
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pone-0010796-g001: Membrane topology of murine FIT2 and FIT1.A and C, FIT2 and FIT1 topological models with N- and C-termini oriented toward the cytosol and six transmembrane domains with a large second luminal loop. The amino acid positions of insertion of FNF tags for the determination of topology by glycosylation site mapping and indirect immunofluorescence are indicated. As indicated, all constructs contain C-terminal V5 and polyhistidine epitope tags. B and D, Increased molecular weights of specific FNF-V5 constructs relative to wild-type FIT2 or FIT1 (indicated by asterisks) detected by Western blot analysis indicated potential glycosylation and luminal orientation. Decreased molecular weight of constructs following PGNaseF treatment indicated glycosylation and luminal orientation. Asterisks indicate glycosylated forms of FIT2 and FIT1 constructs. The multiple asterisks indicated for the FIT2 construct 51 is due to two tandem FNF glycosylation sites inserted into that construct (arrow indicates non-glycosylated construct 51). B and D are representative of two independent experiments.

Mentions: In order to determine the orientation of the N- and C-terminus and the number and orientation of transmembrane domains, we performed epitope tag-glycosylation site mapping, a method that was successfully used to determine the topology of the ER-resident multispanning membrane protein Insig-1 [16]. In this method, a glycosylation consensus motif can be engineered into predicted cytosolic and luminal portions of ER-resident membrane proteins. Only inserted motifs that are exposed to the ER lumen can become accessible to glycosyltransferases in the ER; therefore, glycosylation indicates a luminal orientation. A glycosylation consensus sequence (NGT) flanked by a FLAG tag (DYKDDDDK) on each side (FLAG-NGT-FLAG, abbreviated FNF) was inserted into each of the possible luminal and cytosolic loops of C-terminally V5- and polyhistidine-tagged FIT2 (designated FIT2-V5, Fig. 1A). Each of these constructs was expressed in HEK293 cells and all were functional in terms of the ability to form lipid droplets (Fig. S1). Western blot analysis showed that FNF tags inserted at amino acid positions 51, 129 and 217 resulted in molecular weight shifts greater than the 19 amino acid addition of the FNF tag, likely indicating glycosylated FIT2-V5 (Fig. 1B). Insertions at amino acids 13, 84, 177, and 254 did not result in molecular weight shifts corresponding to greater than 19 amino acids–the molecular weight of the FNF tag (Fig. 1B). To prove that these molecular weight shifts were the consequence of glycosylation, cell lysates were treated with the endoglycosidase PNGaseF in order to cleave N-linked glycans. Lysates from cells expressing FIT2 constructs with FNF insertions at positions 51, 129, and 217 that were treated with PNGase F decreased in molecular weight, consistent with glycosylation and the localization of these domains to the ER lumen. PNGaseF treatment did not affect the apparent molecular weight of FNF insertions at 13, 84, 177, or 254, indicating that these loops are cytosolic (Fig. 1B).


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

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

Membrane topology of murine FIT2 and FIT1.A and C, FIT2 and FIT1 topological models with N- and C-termini oriented toward the cytosol and six transmembrane domains with a large second luminal loop. The amino acid positions of insertion of FNF tags for the determination of topology by glycosylation site mapping and indirect immunofluorescence are indicated. As indicated, all constructs contain C-terminal V5 and polyhistidine epitope tags. B and D, Increased molecular weights of specific FNF-V5 constructs relative to wild-type FIT2 or FIT1 (indicated by asterisks) detected by Western blot analysis indicated potential glycosylation and luminal orientation. Decreased molecular weight of constructs following PGNaseF treatment indicated glycosylation and luminal orientation. Asterisks indicate glycosylated forms of FIT2 and FIT1 constructs. The multiple asterisks indicated for the FIT2 construct 51 is due to two tandem FNF glycosylation sites inserted into that construct (arrow indicates non-glycosylated construct 51). B and D are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875400&req=5

pone-0010796-g001: Membrane topology of murine FIT2 and FIT1.A and C, FIT2 and FIT1 topological models with N- and C-termini oriented toward the cytosol and six transmembrane domains with a large second luminal loop. The amino acid positions of insertion of FNF tags for the determination of topology by glycosylation site mapping and indirect immunofluorescence are indicated. As indicated, all constructs contain C-terminal V5 and polyhistidine epitope tags. B and D, Increased molecular weights of specific FNF-V5 constructs relative to wild-type FIT2 or FIT1 (indicated by asterisks) detected by Western blot analysis indicated potential glycosylation and luminal orientation. Decreased molecular weight of constructs following PGNaseF treatment indicated glycosylation and luminal orientation. Asterisks indicate glycosylated forms of FIT2 and FIT1 constructs. The multiple asterisks indicated for the FIT2 construct 51 is due to two tandem FNF glycosylation sites inserted into that construct (arrow indicates non-glycosylated construct 51). B and D are representative of two independent experiments.
Mentions: In order to determine the orientation of the N- and C-terminus and the number and orientation of transmembrane domains, we performed epitope tag-glycosylation site mapping, a method that was successfully used to determine the topology of the ER-resident multispanning membrane protein Insig-1 [16]. In this method, a glycosylation consensus motif can be engineered into predicted cytosolic and luminal portions of ER-resident membrane proteins. Only inserted motifs that are exposed to the ER lumen can become accessible to glycosyltransferases in the ER; therefore, glycosylation indicates a luminal orientation. A glycosylation consensus sequence (NGT) flanked by a FLAG tag (DYKDDDDK) on each side (FLAG-NGT-FLAG, abbreviated FNF) was inserted into each of the possible luminal and cytosolic loops of C-terminally V5- and polyhistidine-tagged FIT2 (designated FIT2-V5, Fig. 1A). Each of these constructs was expressed in HEK293 cells and all were functional in terms of the ability to form lipid droplets (Fig. S1). Western blot analysis showed that FNF tags inserted at amino acid positions 51, 129 and 217 resulted in molecular weight shifts greater than the 19 amino acid addition of the FNF tag, likely indicating glycosylated FIT2-V5 (Fig. 1B). Insertions at amino acids 13, 84, 177, and 254 did not result in molecular weight shifts corresponding to greater than 19 amino acids–the molecular weight of the FNF tag (Fig. 1B). To prove that these molecular weight shifts were the consequence of glycosylation, cell lysates were treated with the endoglycosidase PNGaseF in order to cleave N-linked glycans. Lysates from cells expressing FIT2 constructs with FNF insertions at positions 51, 129, and 217 that were treated with PNGase F decreased in molecular weight, consistent with glycosylation and the localization of these domains to the ER lumen. PNGaseF treatment did not affect the apparent molecular weight of FNF insertions at 13, 84, 177, or 254, indicating that these loops are cytosolic (Fig. 1B).

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
Related in: MedlinePlus