Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids

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.

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Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes

Biochemical Journal ◽

10.1042/bj3210233 ◽

1997 ◽

Vol 321 (1) ◽

pp. 233-238 ◽

Cited By ~ 14

Author(s):

Eric HAJDUCH ◽

J. Carlos ALEDO ◽

Colin WATTS ◽

Harinder S. HUNDAL

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Glucose Uptake ◽

Glucose Transport ◽

Tetanus Toxin ◽

Glucose Transporter ◽

Detectable Effect ◽

Glut4 Translocation ◽

Rat Adipocytes ◽

Isolated Rat

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.

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Changes in plasma membrane protein structure after photodynamic action in freshly isolated rat pancreatic acini. An FTIR study

Journal of Photochemistry and Photobiology B Biology ◽

10.1016/j.jphotobiol.2003.07.001 ◽

2003 ◽

Vol 71 (1-3) ◽

pp. 27-34 ◽

Cited By ~ 6

Author(s):

Nan Wang ◽

Yuan Liu ◽

Meng Xia Xie ◽

Zong Jie Cui

Keyword(s):

Plasma Membrane ◽

Protein Structure ◽

Membrane Protein ◽

Membrane Protein Structure ◽

Plasma Membrane Protein ◽

Pancreatic Acini ◽

Photodynamic Action ◽

Ftir Study ◽

Isolated Rat

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A review of traditional and emerging methods to characterize lipid–protein interactions in biological membranes

Analytical Methods ◽

10.1039/c5ay00599j ◽

2015 ◽

Vol 7 (17) ◽

pp. 7076-7094 ◽

Cited By ~ 16

Author(s):

Chih-Yun Hsia ◽

Mark J. Richards ◽

Susan Daniel

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Protein Interactions ◽

Protein Structures ◽

Biological Membranes ◽

Biological Functions ◽

Cell Plasma Membrane ◽

Lipid Interactions ◽

Cell Plasma ◽

Lipid Protein Interactions

Lipid–protein interactions are essential for modulating membrane protein structures and biological functions in the cell plasma membrane. In this review we describe the salient features of classical and emerging methodologies for studying protein–lipid interactions and their limitations.

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Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland

Biochemical Journal ◽

10.1042/bj2720099 ◽

1990 ◽

Vol 272 (1) ◽

pp. 99-105 ◽

Cited By ~ 28

Author(s):

R J Madon ◽

S Martin ◽

A Davies ◽

H A C Fawcett ◽

D J Flint ◽

...

Keyword(s):

Plasma Membrane ◽

Mammary Gland ◽

Membrane Protein ◽

Rat Brain ◽

Glucose Transport ◽

Binding Sites ◽

Plasma Membranes ◽

Glucose Transporter ◽

Cytochalasin B ◽

Golgi Vesicle

Plasma membrane- and Golgi vesicle-enriched membrane fractions were prepared from day-10 lactating rat mammary glands. Each fraction was found to contain a single set of D-glucose-inhibitable cytochalasin B-binding sites: plasma membranes and Golgi vesicles bound 20 +/- 2 and 53 +/- 4 pmol of cytochalasin/mg of membrane protein (means +/- S.E.M.), with dissociation constants of 259 +/- 47 and 520 +/- 47 nM respectively. Anti-peptide antibodies against the C-terminal region (residues 477-492) of the rat brain/human erythrocyte glucose transporter labelled a sharp band of apparent Mr 50,000 on Western blots of both fractions. Treatment with endoglycosidase F before blotting decreased the apparent Mr of this band to 38,000, indicating that it corresponded to a glycoprotein. Confirmation that this immunologically cross-reactive band was a glucose transporter was provided by the demonstration that it could be photoaffinity-labelled, in a D-glucose-sensitive fashion, with cytochalasin B. Quantitative Western blotting studies yielded values of 28 +/- 5 and 23 +/- 3 pmol of immunologically cross-reactive glucose transporters/mg of membrane protein in the plasma membrane and Golgi vesicle fractions respectively. From comparison with the concentration of cytochalasin B-binding sites, it is concluded that a protein homologous to the rat brain glucose transporter constitutes the major glucose transport species in the plasma membranes of mammary gland epithelial cells. Glucose transporters are also found in the Golgi membranes of these cells, at least half of them being similar, if not identical, to the transporters of the plasma membrane. However, their function in this location remains unclear.

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Explosion in the complexity of membrane protein recycling

AJP Cell Physiology ◽

10.1152/ajpcell.00171.2020 ◽

2020 ◽

Author(s):

Fiona J McDonald

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Protein ◽

Protein Complexes ◽

Epithelial Polarity ◽

Lipid Interactions

For decades, recycling of membrane proteins has been represented in figures by arrows between the 'endosome' and the plasma membrane, but recently there has been an explosion in the understanding of the mechanisms and protein complexes required to facilitate protein recycling. Here, some key discoveries will be introduced, including assigning function to a number of recently recognized protein complexes, and linking their function to protein recycling. Further, the importance of lipid interactions, and links to diseases and epithelial polarity will be summarized.

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Membrane Protein–Lipid Interactions Probed Using Mass Spectrometry

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-111508 ◽

2019 ◽

Vol 88 (1) ◽

pp. 85-111 ◽

Cited By ~ 35

Author(s):

Jani Reddy Bolla ◽

Mark T. Agasid ◽

Shahid Mehmood ◽

Carol V. Robinson

Keyword(s):

Mass Spectrometry ◽

Membrane Protein ◽

Lipid Bilayers ◽

Protein Interactions ◽

Native Mass Spectrometry ◽

Lipid Interactions ◽

X Ray Crystallography ◽

Native Ms ◽

Molecular Aspects ◽

Lipid Protein Interactions

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid–protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein–lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo–electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein–lipid interactions in the native environment.

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The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e16-08-0586 ◽

2016 ◽

Vol 27 (24) ◽

pp. 3883-3893 ◽

Cited By ~ 26

Author(s):

Yoshiki Tanaka ◽

Natsuki Ono ◽

Takahiro Shima ◽

Gaku Tanaka ◽

Yohei Katoh ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Bilayers ◽

Golgi Complex ◽

Membrane Trafficking ◽

Glucose Transporter ◽

Glucose Transporter 1 ◽

Recycling Endosomes ◽

Type Iv ◽

Late Endosomes ◽

P Type

Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that translocate phospholipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of lipid bilayers. In Saccharomyces cerevisiae, P4-ATPases are localized to specific subcellular compartments and play roles in compartment-mediated membrane trafficking; however, roles of mammalian P4-ATPases in membrane trafficking are poorly understood. We previously reported that ATP9A, one of 14 human P4-ATPases, is localized to endosomal compartments and the Golgi complex. In this study, we found that ATP9A is localized to phosphatidylserine (PS)-positive early and recycling endosomes, but not late endosomes, in HeLa cells. Depletion of ATP9A delayed the recycling of transferrin from endosomes to the plasma membrane, although it did not affect the morphology of endosomal structures. Moreover, depletion of ATP9A caused accumulation of glucose transporter 1 in endosomes, probably by inhibiting their recycling. By contrast, depletion of ATP9A affected neither the early/late endosomal transport and degradation of epidermal growth factor (EGF) nor the transport of Shiga toxin B fragment from early/recycling endosomes to the Golgi complex. Therefore ATP9A plays a crucial role in recycling from endosomes to the plasma membrane.

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