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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02943

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Endocytosis of the glucose transporter GLUT8 is mediated by interaction of a dileucine motif with the ß2-adaptin subunit of the AP-2 adaptor complex

German Institute of Human Nutrition, Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany

View larger version (59K): [in a new window]   Fig. 1. Subcellular distribution of HA-GLUT8 expressed in HeLa cells. Cells were grown on coverslips and either transfected with HA-GLUT8 or HA-GLUT8-LL/AA alone, or co-transfected with HA-GLUT8 and dominant-negative dynamin-K44A, or dynamin-K44A alone. After transfection (48 hours), non-permeabilized (-TX-100) and permeabilized (+TX-100) cells were stained for the HA-epitope tag and analyzed by confocal laser scanning microscopy. Bars, 20 µm.

View larger version (14K): [in a new window]   Fig. 2. Cell surface expression of HA-GLUT8 in HeLa cells. Cells were grown in six-well plates and transfected with HA-GLUT8 without or with plasmid for dynamin K44A, and with mutant HA-GLUT8-LL/AA. After 48 hours, cells were harvested and the expression of the HA-GLUTs was determined by western blotting using an anti-HA antibody. In parallel, cell-surface levels of the HA-GLUTs were determined using an antibody binding assay as described in the Materials and Methods. The cell surface-associated radioactivity was normalized to the relative protein expression level of each respective mutant. Results are the means ± s.d. of a representative experiment performed in duplicate.

View larger version (25K): [in a new window]   Fig. 3. Interaction of the N-terminus of GLUT8 with ß-adaptins. (A) Yeast cells (strain SFY526) co-transformed with plasmids for GAL4-BD/GLUT8-NTs and GAL4-AD/adaptins from AP-2 were grown to mid-log phase, harvested and assayed for ß-galactosidase activity using CPRG as substrate (see the Materials and Methods). (B) ß-galactosidase activity of yeast cells co-expressing GAL4-BD/GLUT8-NTs and different GAL4-AD/ß-adaptins. (C) Immobilized GST fusion proteins of the GLUT8 N-terminus, wild-type GST-GLUT8-NT and the LL/AA mutant, were incubated with HeLa cell lysates (overnight, 4°C). After washing, bound AP-2 was analyzed by SDS-PAGE and western blotting for -adaptin. (D) Full-length ß1-adaptin or ß2-adaptin was in vitro translated in the presence of [35S]methionine and incubated with 15 µg of immobilized wild-type GST-GLUT8-NT or LL/AA mutant (1 hour, 4°C). After washing, bound adaptins were analyzed by SDS-PAGE and autoradiography. (E) (Top) Structural domains of human ß2-adaptin and structure of truncation mutants of ß2-adaptin. (Bottom) Yeast two-hybrid analyses of GAL4-BD/GLUT8-NT and trunk-hinge (M1-S726) and hinge-ear (I588-N937) truncation mutants of ß2-adaptin fused to GAL4-AD. C and D are representative of a total of seven independent experiments, all other results are the means ± s.e.m. of at least three independent experiments performed in duplicates.

View larger version (32K): [in a new window]   Fig. 4. RNAi-mediated knockdown of µ2-adaptin in HeLa cells. Cells were transfected with a plasmid construct for the µ2 siRNA (pSuper/µ2-1) or with empty pSuper vector (con). After 6 days in cell culture, the cells were harvested and lysed. Then, the proteins (20 µg/lane) were subjected to SDS-PAGE, and the expression of adaptins in the crude lysates was determined by western blotting using antibodies against -adaptin (112 kDa), ß-adaptin (106 kDa), -adaptin (104 kDa), µ2-adaptin (50 kDa), and -tubulin as described in the Materials and Methods.

View larger version (12K): [in a new window]   Fig. 5. 125I-transferrin uptake in AP-2-depleted HeLa cells. Cells were transfected with empty pSuper vector (Control) or pSuper/µ2-1 construct, and cultured for 6 days. Then, the cells were assayed for uptake of 125I-labeled transferrin at 37°C for 15 minutes as described in the Materials and Methods, and the incorporated radioactivity was quantified using a -counter. Results are the means ± s.d. of quadruplicate samples from one of two experiments.

View larger version (25K): [in a new window]   Fig. 6. Subcellular distribution and cell-surface targeting of HA-GLUT8 in AP-2-depleted HeLa cells. (A) Cells were grown on coverslips and co-transfected with HA-GLUT8 and empty pSuper vector, HA-GLUT8 and pSuper/µ2-1, and HA-GLUT8-LL/AA and empty pSuper vector. After 6 days in culture, non-permeabilized cells were stained for the HA-epitope tag and analyzed by confocal laser scanning microscopy as described in the Materials and Methods. Bars, 20 µm. (B) Cells were co-transfected with HA-GLUT8s and empty pSuper vector (-) or pSuper/µ2-1 construct (+), and cultured for 6 days. Then, the cell-surface levels of the HA-GLUTs were determined using an antibody binding assay as described in the Materials and Methods. In parallel, the expression of the HA-GLUTs was determined by western blot using an anti-HA antibody (inset). The cell surface-associated radioactivity was normalized to the level of the HA-GLUT8-LL/AA mutant. Results are the means ± s.e.m. of four to six independent experiments performed in duplicate. *P<0.05 (paired Student's t-test) comparing pSuper/µ2-1 cells and control cells.

View larger version (18K): [in a new window]   Fig. 7. Yeast two-hybrid interaction of mutant GLUT8-NTs with adaptins from AP-2. (A) Amino acid sequence of GLUT8-NT constructs. (B) Yeast two-hybrid analyses of the GAL4-BD/GLUT8-NTs and GAL4-AD/adaptins from AP-2. Results are the means ± s.e.m. of three independent experiments performed in duplicate.

View larger version (19K): [in a new window]   Fig. 8. Cell surface targeting of HA-GLUT8 and YTRF mutant in HeLa cells. Cells were transfected with plasmids for HA-GLUT8, LL/AA and YTRF mutant, and cultured for 4-6 days. The expression of the HA-GLUTs was determined by western blot using an anti-HA antibody (inset). In parallel, cell-surface levels of the HA-GLUTs were determined using an antibody binding assay as described in the Materials and Methods. Results are the means ± s.d. of three independent experiment performed in duplicate to quadruplicate.

View larger version (93K): [in a new window]   Fig. 9. Co-staining of HA-GLUT8, HA-GLUT8/YTRF and transferrin in HeLa cells. Cells were grown on coverslips and either transfected with HA-GLUT8 or HA-GLUT8/YTRF. After 3 days in culture, serum-starved cells were incubated with transferrin (Tf) conjugated to Alexa Fluor 633 for 30 minutes at 37°C, thoroughly washed with PBS and fixed in 4% paraformaldehyde. Then the cells were stained for the HA-epitope tag and analyzed by confocal laser scanning microscopy as described in the Materials and Methods Squares indicate magnified regions (mag.). Bars, 20 µm.

View larger version (153K): [in a new window]   Fig. 10. Steady-state distribution of HA-GLUT8s within the endosomal/lysosomal pathway and the Golgi apparatus. HeLa cells were transfected with HA-GLUT8 and cultured for 3 days. Then, the cells were stained with either Bodipy-TR ceramide or LysoTracker-Red, or stained for AP-1 or AP-2 using a monoclonal antibody against - or µ2-adaptin as described in the Materials and Methods. After co-staining for the HA-epitope tag, the cells and analyzed by confocal laser scanning microscopy. No difference in the staining pattern was observed for HA-GLUT8 and HA-GLUT8/YTRF (data not shown). Squares indicate magnified regions (mag.). Bars, 20 µm.