AP-1A–HA, but not AP-1B-HA, colocalizes with furin. LLC-PK1 cells transfected with μ1A-HA (top) or μ1B-HA (bottom) were double-labeled with anti-HA antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488–labeled (anti-HA staining) and Alexa 594–labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy. Representative images are shown (merged images are shown on right).

AP-1–HA is found on clathrin-coated structures. LLC-PK1::μ1A-HA (A) or LLC-PK1::μ1B-HA transfectants (B) were analyzed by immunoelectron microscopy (see Materials and Methods for details). HA-tagged μ1 proteins were visualized using anti-HA antibodies, followed by incubations with anti–mouse IgG antibodies and protein A–gold. Arrowheads highlight gold particles and arrows denote clathrin-coated structures. Bar, 100 nm.

AP-1A is necessary for furin localization at the TGN. μ1A^−/− fibroblasts stably transfected with μ1B alone (top) or with μ1B and μ1A-HA (bottom) were double-labeled with anti–γ-adaptin antibodies (left) and antifurin antibodies (middle) in combination with Alexa 488–labeled (anti-γ staining) and Alexa 594–labeled (antifurin staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown. The arrows denote regions in the AP-1B/AP-1A–HA transfectants that are positive for AP-1 but negative for furin.

AP-1–HA localizes to a perinuclear region. LLC-PK1::μ1A-HA transfectants (A–C, top) and LLC-PK1 cells transfected with μ1B-HA (bottom) were fixed and incubated with anti-HA antibodies (A, left) in combination with (A) anti–γ-adaptin antibodies (middle), (B) anti-GM130 antibodies, or (C) Texas red–labeled Tfn. These incubations were followed by an incubation with Alexa 488–labeled (anti-HA staining) and Alexa 594–labeled (anti–γ-adaptin and anti-GM130 staining) secondary antibodies. Specimens were analyzed by confocal microscopy and representative images are shown.

AP-1B directly interacts with LDLR. (A) LLC-PK1::μ1A or LLC-PK1::μ1B transfectants were infected with defective adenoviruses encoding the LDLR. 1 d after the infections the cells were lysed and the LDLR was immunoprecipitated (IP) with anti-LDLR antibody (see Materials and Methods for details). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting. LDLR, γ-adaptin, and μ1B were detected by immunodecoration with anti-LDLR antibodies, anti–γ-adaptin antibodies, or specific anti-μ1B antibodies raised against the COOH terminus of μ1B, respectively. Samples representing equivalent amounts of starting cell protein were added to each lane. (B) LLC-PK1 cells transfected with μ1B (lanes 1–4) or μ1B-HA (lanes 5 and 6) were infected with adLDLR. 1 d after infection the cells were incubated with the cross-linker DTSSP (100 μM). After quenching, the cells were lysed and the LDLR was immunoprecipitated as in A (details are described in Materials and Methods). Immunoprecipitates were analyzed as described in A. Clathrin heavy chain was detected by immunodecoration with anticlathrin heavy chain antibodies. (C) LLC-PK1::μ1B transfectants were infected with adLDLR (lanes 1 and 2) or adLDLR(Y18A/G34D) (lanes 3 and 4) and analyzed as described in B.

Expression and function of m1-HA. (A) Diagram showing the position of the internal HA-tag in μ1 protein (423 amino acids total). (B) Nontransfected LLC-PK1 cells, as well as LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants, were lysed in RIPA buffer and 5 and 10 μg of total protein were analyzed by Western blotting. γ-Adaptin, μ1-HA, and μ1 proteins were detected by immunodecoration with anti–γ-adaptin antibodies, anti-HA antibodies, and an antibody that cross-reacts with μ1A and μ1B, respectively. (C) LLC-PK1::μ1A-HA and LLC-PK1::μ1B-HA transfectants were metabolically labeled with [³⁵S]methionine/cysteine overnight and lysed in Triton X-100 buffer. AP-1 complexes were immunoprecipitated using anti–γ-adaptin. The immunoprecipitates were boiled in SDS and one half of the extract was directly subjected to SDS-PAGE analysis. The remaining extract was diluted 20-fold in lysis buffer and μ1A-HA or μ1B-HA proteins were recaptured using anti-HA antibodies or specific anti-μ1B antibodies directed against the COOH terminus of μ1B, respectively. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. (D) LLC-PK1::μ1A-HA (top) and LLC-PK1::μ1B-HA (bottom) transfectants were grown on Transwell filters and infected with recombinant adenoviruses encoding the FcLR(CT22) chimera (left) or the LDLR (right). 2 d after the infection viable cells were incubated with antibodies directed against the ectodomain of FcLR or LDLR, respectively, fixed, and incubated with Alexa 488–labeled secondary antibodies as described in Material and Methods. Specimens were analyzed by confocal microscopy. Representative X–Z sections are shown.