Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network

Abstract

Autophagy is an intracellular bulk protein degradation system. Beclin is known to be involved in this process; however, its role is unclear. In this study, we showed that Beclin was co-immunoprecipitated with phosphatidylinositol (PtdIns) 3-kinase, which is also required for autophagy, suggesting that Beclin is a component of the PtdIns 3-kinase complex. Quantitative analyses using a cross-linker showed that all Beclin forms a complex with PtdIns 3-kinase, whereas approximately 50% of PtdIns 3-kinase remains free from Beclin. Indirect immunofluorescence microscopy demonstrated that the majority of Beclin and PtdIns 3-kinase localize to the trans-Golgi network (TGN). Some PtdIns 3-kinase is also distributed in the late endosome. These results suggest that Beclin and PtdIns 3-kinase control autophagy as a complex at the TGN.

Fig. 1. Beclin and PtdIns 3-kinase form a complex. (A) Cell lysates prepared from HeLa cells were solubilized with Triton X-100 and subjected to immunoprecipitation using preimmune sera (lane 1) or anti-Beclin antibodies (lanes 2 and 3). The immunoprecipitates were incubated with phosphatidylinositol, [γ-³²P]ATP and 60 µM cold ATP in the presence of Mn²⁺ (lanes 1 and 2) or Mg²⁺ (lane 3) for 5 min at 30°C. (B) Immunoprecipitates of anti-Beclin antibodies prepared as described in (A) were assayed for PI 3-kinase activity in the presence of wortmannin at concentrations of 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4). The labeled lipids were extracted and separated by thin layer chromatography, followed by detection by autoradiography using a bioimaging analyzer BAS2000 (Fuji Film). (C) Cell lysates prepared from HeLa cells were solubilized with Triton X-100 and incubated with protein A-immobilized anti-Beclin (lane 2) or anti-PtdIns 3-kinase antibodies (lane 4). As controls, preimmune sera of the respective antibodies (lanes 1 and 3) were used. Retained proteins were separated by SDS–PAGE and detected by immunoblotting with anti-PtdIns 3-kinase (upper panels) and anti-Beclin (lower panels) antibodies.

Fig. 2. Cross-linking of Beclin and PtdIns 3-kinase. (A) Total cell lysates were treated with DSP (lanes 3, 4, 7 and 8) or its solvent, dimethylsulfoxide (lanes 1, 2, 5 and 6), at 4°C for 2 h. Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A-immobilized anti-PtdIns 3-kinase (PtdIns 3-K) (lanes 1–4) or Beclin (lanes 5–8) antibodies. Immunoprecipitates were eluted with 1× SDS sample buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, a trace amount of Bromophenol blue). After treatment with 5% 2-mercaptoethanol, proteins were separated by SDS–PAGE, followed by detection by immunoblotting with anti-Beclin (upper panels) and anti-PtdIns 3-kinase (lower panels) antibodies. (B) Cell lysates prepared from HeLa cells were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h. The membrane (lanes 1 and 3) and supernatant (lanes 2 and 4) fractions were subjected to SDS–PAGE, followed by immunoblotting with anti-PtdIns 3-kinase (lanes 1 and 2) and anti-Beclin (lanes 3 and 4) antibodies. (C) Membrane (lanes 1–4; 100 000 g, 30 min, pellet) and soluble fractions (lanes 5–8; 100 000 g, 30 min, supernatant) prepared from HeLa cells were treated with DSP (lanes 3, 4, 7 and 8) or dimethylsulfoxide (lanes 1, 2, 5 and 6). Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A-immobilized anti-PtdIns 3-kinase antibodies. Immunoprecipitates were separated by SDS–PAGE under a reducing condition, followed by detection by immunoblotting with Beclin antibodies. F and E indicate flow-through and elute fracions, respectively.

Fig. 3. Beclin localizes to the TGN. HeLa (A, D, E and F), BNL CL.2 (B) and H-4-II-E cells (C) were grown on cover slips. Cells were permeabilized with digitonin before (C, D and E) or after (A, B and F) fixation with 3% paraformaldehyde. Cells were double-labeled with anti-Beclin (A–F, left panels) and either anti-syntaxin 6 (A and B, middle panels), anti-TGN38 (C, middle panel), anti-EEA1 (D, middle panel), anti-LBPA (E, middle panel) or anti-TfR (F, middle panel) antibodies. In the merged images (right panels), yellow indicates co-localization. Bar, 10 µm.

Fig. 4. Immunofluorescence localization of PtdIns 3-kinase. HeLa (A and C) and H-4-II-E cells (B) were permeabilized, fixed, and then subjected to immunostaining. Cells were double-labeled with anti-PtdIns 3-kinase (A–C, left panels) and either anti-syntaxin 6 (A, middle panel), anti-TGN38 (B, middle panel) or anti-LBPA (C, middle panel) antibodies. Bar, 10 µm.