FIGURE 1.

APP specifically recruits Mint3 to the Golgi, in an Arf-dependent fashion. A, cells were transfected with empty vector (pFUGW) or pFUGW-APP as indicated. Cells were fixed 1 day later and stained with antibodies against the C terminus of APP and γ-adaptin (AP-1), ϵ4 (AP-4), or Mint3. Maximum intensity projections of wide field images are shown. B, cells were transfected with APP or CD8-APP, as indicated, and stained with antibodies to APP and GM130 (top panels) or CD8 and giantin (bottom panels). Isosurfaces were built based on GM130 (top panels) or giantin (bottom panels) staining, and the total intensity of APP (top panels) or CD8 (bottom panels) within the isosurface was calculated. A heat map of intensities ranging from 0 to 65,536 was generated (color bar on right). C, mock-transfected cells or cells expressing CD8-APP were treated as described in B and stained with antibodies against Mint3 and giantin. Wide field stacks of images were collected, deconvolved, and imported into Imaris where isosurfaces were built based on giantin staining; the total amount of Mint3 within the isosurface was recorded and expressed as a ratio of intensity/giantin volume (intensity/μm³ × 10⁻³). Values were normalized to control cells maintained at 37 °C. Statistical significance is indicated with #, p < 0.01, and *, p < 0.05, when analyzed using ANOVA. D, APP-dependent Mint3 recruitment is BFA-sensitive and rapidly reversible. HeLaM cells expressing APP were treated with either vehicle or BFA for 2 min (see “Experimental Procedures”). Fresh, pre-warmed medium was then used to wash out the drug, and cells were allowed to recover for the times indicated, before fixation and staining with antibodies against Mint3 and giantin. Confocal images are shown.