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Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids *

View Article: PubMed Central - PubMed

ABSTRACT

The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.

No MeSH data available.


GLUT4 is randomly inserted into liposomes. Western blotting analysis of FLAG-GLUT4 using anti-FLAG antibody before and after TEV protease cleavage of an engineered N-terminal FLAG tag. GLUT4 cleavage was carried out in LMNG detergent micelles or in 70% egg PC, 15% POPA, 15% POPE liposomes for 0, 3, and 24 h. FLAG-GLUT4 (sum of both monomer and dimer) was quantified using an Odyssey Infrared Imaging System.
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Figure 2: GLUT4 is randomly inserted into liposomes. Western blotting analysis of FLAG-GLUT4 using anti-FLAG antibody before and after TEV protease cleavage of an engineered N-terminal FLAG tag. GLUT4 cleavage was carried out in LMNG detergent micelles or in 70% egg PC, 15% POPA, 15% POPE liposomes for 0, 3, and 24 h. FLAG-GLUT4 (sum of both monomer and dimer) was quantified using an Odyssey Infrared Imaging System.

Mentions: To study the effect of different phospholipids at specific concentrations on the activity of GLUT4 and GLUT3, we developed an assay system allowing us to measure uptake of radiolabeled glucose into liposomes that contained purified transporter embedded in a tightly controlled lipid environment. Both transporters showed saturable uptake over seconds to minutes of their primary substrate d-glucose over the non-transportable l-glucose in liposomes containing 70% egg phosphatidylcholine (egg PC), 15% phosphatidic acid (PA), and 15% PE (mol/mol) (Fig. 1). Uptake was normalized to the amount of transporter in each uptake assay as described under “Experimental Procedures.” Transport could be completely abolished using the GLUT-specific inhibitor cytochalasin B (Fig. 1a). As expected for random insertion, the transporter incorporated into liposomes with roughly half in an outward-facing and half in an inward-facing orientation. Evidence for this conclusion was obtained from Western blotting analysis of the transporter using anti-FLAG antibody before and after tobacco etch virus (TEV) protease cleavage of an engineered N-terminal FLAG tag (Fig. 2). In detergent micelles, the protease has access to both faces of the transporter, although in liposomes, it can only access transporters that orient with their N terminus facing outward. We determined cleavage of the FLAG tag is ∼100% in micelles, whereas only ∼50% of all transporters in liposomes are cleaved by TEV protease. Interestingly, the immunoblot revealed that a fraction of GLUT4 in both detergent and liposomes ran as a dimer when tagged with FLAG but as a monomer when cleaved. All subsequent liposome experiments were carried out using tag-cleaved GLUT4 or GLUT3.


Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids *
GLUT4 is randomly inserted into liposomes. Western blotting analysis of FLAG-GLUT4 using anti-FLAG antibody before and after TEV protease cleavage of an engineered N-terminal FLAG tag. GLUT4 cleavage was carried out in LMNG detergent micelles or in 70% egg PC, 15% POPA, 15% POPE liposomes for 0, 3, and 24 h. FLAG-GLUT4 (sum of both monomer and dimer) was quantified using an Odyssey Infrared Imaging System.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016126&req=5

Figure 2: GLUT4 is randomly inserted into liposomes. Western blotting analysis of FLAG-GLUT4 using anti-FLAG antibody before and after TEV protease cleavage of an engineered N-terminal FLAG tag. GLUT4 cleavage was carried out in LMNG detergent micelles or in 70% egg PC, 15% POPA, 15% POPE liposomes for 0, 3, and 24 h. FLAG-GLUT4 (sum of both monomer and dimer) was quantified using an Odyssey Infrared Imaging System.
Mentions: To study the effect of different phospholipids at specific concentrations on the activity of GLUT4 and GLUT3, we developed an assay system allowing us to measure uptake of radiolabeled glucose into liposomes that contained purified transporter embedded in a tightly controlled lipid environment. Both transporters showed saturable uptake over seconds to minutes of their primary substrate d-glucose over the non-transportable l-glucose in liposomes containing 70% egg phosphatidylcholine (egg PC), 15% phosphatidic acid (PA), and 15% PE (mol/mol) (Fig. 1). Uptake was normalized to the amount of transporter in each uptake assay as described under “Experimental Procedures.” Transport could be completely abolished using the GLUT-specific inhibitor cytochalasin B (Fig. 1a). As expected for random insertion, the transporter incorporated into liposomes with roughly half in an outward-facing and half in an inward-facing orientation. Evidence for this conclusion was obtained from Western blotting analysis of the transporter using anti-FLAG antibody before and after tobacco etch virus (TEV) protease cleavage of an engineered N-terminal FLAG tag (Fig. 2). In detergent micelles, the protease has access to both faces of the transporter, although in liposomes, it can only access transporters that orient with their N terminus facing outward. We determined cleavage of the FLAG tag is ∼100% in micelles, whereas only ∼50% of all transporters in liposomes are cleaved by TEV protease. Interestingly, the immunoblot revealed that a fraction of GLUT4 in both detergent and liposomes ran as a dimer when tagged with FLAG but as a monomer when cleaved. All subsequent liposome experiments were carried out using tag-cleaved GLUT4 or GLUT3.

View Article: PubMed Central - PubMed

ABSTRACT

The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.

No MeSH data available.