QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schmidt, U. Articles by Al-Hasani, H. Search for Related Content PubMed PubMed Citation Articles by Schmidt, U. Articles by Al-Hasani, H. Social Bookmarking                         What's this?

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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter