FIG. 4.

Tsg101 multimerization mediated by sequences in the leucine zipper and C-terminal domains. (A) 293T cells were transfected with a Myc-VPS28 expression plasmid alone (lanes −) or with both Tsg101-pA and Myc-VPS28 expression plasmids (lanes +). Cytoplasmic lysates (left panels) and IgG-bound protein complexes (right panels) were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting (WB) with anti-Tsg101 (α-Tsg101) (top and middle panels) or anti-Myc (lower panels) antibodies. (B) Yeast two-hybrid assay of Tsg101 multimerization. Y190 cells were transformed with GAL4-Tsg10(1-390) (full length) or GAL4-Tsg10(1-303) expression plasmids along with a plasmid expressing a full-length or deletion mutant VP16-Tsg101 expression plasmid, as indicated. The mean level of β-galactosidase expression (± standard deviation) in three pools of transformants is shown. (C) Coprecipitation of YFP-Tsg101 and Tsg101-pA. 293T cells were transfected with a YFP-Tsg101 (left panels) or a YFP-Tsg101(303-360) (right panels) expression plasmid in the presence (lanes +) or absence (lanes −) of a Tsg101-pA expression plasmid. Cell lysates (top panels) or IgG-bound protein complexes (bottom panels) were analyzed by Western blotting with an anti-GFP antibody. (D) Yeast two-hybrid analysis of mutant Tsg101 protein multimerization. Y190 cells were transformed with GAL4-Tsg101 along with a plasmid expressing a full-length or mutant VP16-Tsg101. The mean level of β-galactosidase expression (± standard deviation) in three pools of transformants is shown. (E) 293T cells were transfected with full-length YFP-Tsg101 (wild type [WT]) or YFP-Tsg101(EP4) and VPS28-pA expression plasmids. Cell lysates (top panel) or IgG-bound protein complexes (bottom panel) were analyzed by Western blotting with an anti-GFP antibody.