RESUMO
Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced green fluorescent protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5-0.7% (w/w) of the total protein, i.e. over 50-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk.
Assuntos
Metabolismo dos Lipídeos , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Animais , Butirofilinas , Bovinos , Membrana Celular/metabolismo , Feminino , Lactação , Metabolismo dos Lipídeos/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/metabolismoRESUMO
Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Fusão de Membrana , Músculo Esquelético/metabolismo , Animais , Vesículas Citoplasmáticas/metabolismo , Exocitose , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 4/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemaglutininas/genética , Hemaglutininas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Músculo Esquelético/citologia , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Sarcolema/metabolismoRESUMO
The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (DeltaPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent Km value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in Km and for a subsequent 150-fold increase in the catalytic efficiency (k(cat)/Km) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.
Assuntos
Glutationa Transferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Baculoviridae/genética , Clonagem Molecular , Dimerização , Ativação Enzimática , Humanos , Cinética , Ácido Okadáico/farmacologia , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de ProteínaRESUMO
Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.
Assuntos
Adipócitos/metabolismo , Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fusão de Membrana/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células 3T3-L1 , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Membrana Celular/genética , Células Cultivadas , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Transportador de Glucose Tipo 4/genética , Hipoglicemiantes/metabolismo , Insulina/metabolismo , Masculino , Fusão de Membrana/genética , Camundongos , Mutação , Ratos , Vesículas Secretórias/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
GLUT1 has recently been suggested to be a binding receptor for human T-cell leukemia virus type 1 (HTLV-1). We used a novel, short-term assay to define the role of GLUT1 in cell-to-cell transmission. Although increasing cell surface levels of GLUT1 enhanced HTLV-I transfer, efficient virus spread correlated largely with heparan sulfate proteoglycan (HSPG) expression on target cells. Moreover, since activated CD4+ T cells and cord blood lymphocytes that are susceptible to HTLV-1 infection expressed undetectable levels of surface GLUT1, these results indicate that GLUT1 and HSPGs are important for efficient cell-to-cell transmission of HTLV-1 but raise concerns on the role of GLUT1 as the HTLV-1 primary binding receptor.
Assuntos
Transportador de Glucose Tipo 1/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Receptores Virais/metabolismo , Vírion/fisiologia , Infecções por HTLV-I/transmissão , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Leucemia de Células T/fisiopatologia , Leucemia de Células T/virologiaRESUMO
To identify novel regulatory components involved in the recycling of the insulin-responsive glucose transporter GLUT4, we have used the yeast two-hybrid system to isolate GLUT4-binding proteins from a rat adipose cell cDNA library. We found a 49-kDa protein (p49/STRAP) that specifically interacts with an acidic amino acid motif (Q7IGSEDG) in the N-terminus of GLUT4. Confocal immunofluorescence microscopy of primary rat adipose cells shows co-localization of myc-p49 with GLUT4 and also with the ER-resident protein calnexin. Insulin stimulation had no effect on GLUT4-binding and subcellular distribution of p49 in adipose cells. However, overexpression of the GLUT4-binding domain of p49 in adipose cells reduces protein synthesis and cell-surface expression of GLUT4, but not of GLUT8. Moreover, cell-surface expression of a p49-binding-deficient GLUT4 mutant (ED/QN) is also reduced. Kinetic analysis of HA-epitope-tagged GLUT4 protein synthesis indicates a possible role of p49 in biosynthesis and/or processing of GLUT4 in adipose cells.
Assuntos
Tecido Adiposo/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Fatores de Transcrição/metabolismo , Tecido Adiposo/citologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Núcleo Celular/química , Transportador de Glucose Tipo 4/análise , Transportador de Glucose Tipo 4/genética , Humanos , Insulina/farmacologia , Camundongos , Dados de Sequência Molecular , Mutação , Mapeamento de Interação de Proteínas , Ratos , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
Studies using adherent cell lines have shown that glucose transporter-1 (GLUT-1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4(+) T cells, heparan sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT-1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4(+) T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8(+) T cells. Conversely, CD8(+) T cells, the primary target of HTLV-2, expressed GLUT-1 at dramatically higher levels than CD4(+) T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8(+) T cells while HTLV-1 SU binding and viral entry were higher on CD4(+) T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4(+) T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT-1 in CD4(+) T cells increased HTLV-2 entry, while expression of HSPGs on CD8(+) T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4(+) and CD8(+) T cells involved in entry.