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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.

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