Interaction of the bovine papillomavirus E6 protein with the clathrin adaptor complex AP-1

Abstract

The E6 gene of the bovine papillomavirus type 1 (BPV-1) is expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major oncoproteins of BPV-1. In this study, we demonstrate an interaction between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex. AP-1 is a four-subunit protein complex required for clathrin-mediated cellular transport from the TGN. The AP-1/E6 interaction was observed in vitro and in cells. The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the gamma subunit. BPV-1 E6 preferentially associated with membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6 was further shown to be recruited to isolated Golgi membranes and to copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1 was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1 to Golgi membranes involves a common factor. Taken together, our results suggest that cytosolic BPV-1 E6 is first recruited to the TGN, where it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived clathrin-coated vesicles. We propose that BPV-1 E6, through its interaction with AP-1, can affect cellular processes involving clathrin-mediated trafficking pathway.

FIG. 1

(A) Schematic of the BPV-1 E6 protein structure. The E6 protein has four Cys-X-X-Cys motifs and is predicted to form two zinc-binding sites (41). X_n indicates the number of residues between the cysteines. The E6 mutants used in this study are also indicated. (B) Identification of BPV-1 E6-associated proteins. ³⁵S-labeled cell lysates from mouse C127 cells were incubated with wild-type (wt) and various mutant GST-E6 fusion proteins. The bound cellular proteins were separated by SDS-PAGE and visualized by autoradiography. The transforming activity (TF) of each E6 mutant and the positions of size standards (in kilodaltons) and of p110, p100, and p50 are indicated. na, not applicable.

FIG. 2

In vitro interaction between BPV-1 E6 and the AP-1 complex. AP-1 and AP-2 complexes purified from bovine brain were incubated with various GST-E6 fusion proteins. The AP complexes were detected by immunoblotting with monoclonal antibody 9A against the β subunit. wt, wild type.

FIG. 3

Interaction between BPV-1 E6 and AP-1 in cells. Cos cells were transiently transfected with vector, FLAG-tagged wild-type (wt) E6, mutant E6 (Δ134-137), or mutant E6 (H105D) and labeled with [³⁵S]methionine-cysteine. Cell lysates were immunoprecipitated for the AP-1 and AP-2 complexes by using monoclonal antibody 9A against the β subunit and for E6 by using the FLAG antibody. The positions of each AP subunit, paxillin, and E6 are indicated.

FIG. 4

Interaction between BPV-1 E6 and the AP-1 subunits. Each of the four subunit of AP-1 was ³⁵S labeled by in vitro translation and incubated with wild-type GST-E6 (wt E6) and mutant GST-E6 (H105D) fusion proteins. The bound proteins were separated by SDS-PAGE and visualized by autoradiography. Sizes are indicated in kilodaltons.

FIG. 5

Mapping of the E6 binding domain on AP-1. AP-1 and AP-2 proteins purified from bovine brain were partially digested by serially diluted trypsin, separated by SDS-PAGE, immobilized on a PVDF membrane, and probed by ³²P labeled GST-E6.

FIG. 6

Study of the interaction between BPV-1 E6 and AP-1 by cell fractionation. (A) Lysates from Cos cells transiently transfected with wild-type (wt) E6 or mutant E6 (C90S) were fractionated into crude cytosolic (a), membrane (b), and nuclear (c) fractions. Each fraction was analyzed by immunoprecipitation (IP) and immunoblotting as indicated. (B) The crude cytosolic fraction from panel A was further subjected to centrifugation at 100,000 × g for 1 h to generate a high-speed cytosol which was immunoprecipitated for AP-1 by using an antibody against the β subunit and for E6 by using the anti-FLAG antibody M2. The immunocomplexes were then assayed by immunoblotting with an antibody against the γ subunit of AP-1 and the anti-FLAG antibody M2 for E6.

FIG. 7

Assembly of BPV-1 E6 into CCV. (A) CCV were isolated from Cos cells transfected with wild-type (wt) or mutant E6 proteins (C90S). The distribution of AP-1 and E6 was determined by immunoblotting. (B) CCV purified from bovine brain were incubated with ³⁵S-labeled, in vitro-translated E6 and immunoprecipitated for clathrin heavy chain by using monoclonal antibody X-22 (3). AP-1 was detected by immunoblotting against the γ subunit, and E6 was detected by autoradiography.

FIG. 8

Recruitment of BPV-1 E6 to Golgi membranes. (A) Isolated rat Golgi membrane (mem), GTPγS, brefeldin A (BFA), and crude cytosol from Cos cells transfected with wild-type (wt) E6 were mixed as indicated. The membrane-bound AP-1 and E6 proteins were pelleted by centrifugation and detected by immunoblotting. (B) High-speed cytosols from Cos cells expressing wild-type E6 or mutant E6 (C90S) were used in the membrane binding assay; 2 μg of AP-1 purified from bovine brain was added to the reaction mixture where indicated.