Role of adaptor-binding motifs in export and segregation of endolysosomal proteins at the Golgi complex. (a) Sequences from the cytosolic domains of TfR, LAMP1, and CD-MPR. Motifs that bind to AP complexes in each protein are highlighted in red. Mutations are indicated with blue letters. (b and c) HeLa cells coexpressing streptavidin–KDEL together with TfR and LAMP1 reporter constructs having mutations in AP-binding motifs, namely TfR-Y20A/GDNS^31-34AAAA (b) or LAMP1-Y404A (c), were imaged live by spinning-disk confocal microscopy. Images are single frames from Video 9. The times after addition of biotin are indicated. (Right) Magnifications of the boxed regions. Bars: (low magnification) 5 µm; (high magnification) 1 µm. (d and e) HeLa cells coexpressing streptavidin–KDEL together with GGA1–GFP and CD-MPR (d) or CD-MPR-L274A/L275A (e) reporter proteins were fixed 30 min after the addition of biotin and imaged by Airyscan microscopy. Bar, 2 µm. Arrows indicate carriers containing GGA1–GFP. (f) Airyscan microscopy of HeLa cells coexpressing streptavidin–KDEL together with CD-MPR-L274A/L275A and TfR-Y20A/GDNS^31-34AAAA reporter proteins 30 min after the addition of biotin. The inset shows magnified images of the boxed regions. Bars, 1 µm.