Function of the R43L and R280C transporters. (A-C) Uptake of C12-NBD-phospholipids PC and PE into ABCA3 vesicles in A549 cells transfected with pUB6/HA-ABCA3 vectors was studied by confocal microscopy. Transfected cells were incubated for 2 h with (A) C12-NBD-PC and (B) C12-NBD-PE containing liposomes (150 μM of NBD-lipid). HA-tag was used to detect ABCA3 WT and R43L, R280C and L101P by immunofluorescence (red). Both C12-NBD-PC and C12-NBD-PE fluorescence (green) frequently colocalized with the ring-like ABCA3 WT signal as well as within the ABCA3-WT vesicles and almost never with the R43L and R280C vesicles. In small sections and in (C) uptake into ABCA3-WT vesicles of both NBD-lipids PC and PE is depicted closer. Uptake through the plasma membrane into the cytoplasm of L101P transfected cells with no functional exogenous ABCA3 was observed as well as numerous cytoplasmic NBD-positive liposome-like vesicles in all A549 cells independent of the ABCA3 mutation shown in (A) and (B). (D) Influence of WT ABCA3 and three mutations on biogenesis of LAMP3+ vesicles in A549 transfected with pUB6/HA-ABCA3. WT ABCA3 (green) induced biogenesis of LAMP3+ vesicles (red) increasing their number and size in A549 cells, while R43L, R280C and L101P proteins showed no such effect (the same was observed in A549 pEYFP-N1/ABCA3 transfected cells - not shown). Representative pictures are shown; scale bars: 5 μm.