PMC

Schematic representation of the ABCA3 and ABCA1 expression constructs used in this study. Twenty-seven different fusion constructs tagged with either an EGFP, a DsRed, or FLAG reporter were used. SP-C isoforms were deliberately tagged at the N terminus to follow the protein to its terminal destination since proSP-C is cleaved at the distal C terminus at or near residue 145 in early post-Golgi compartments in a non-cell-specific manner.

The LLLWKN motif targets proteins to post-Golgi vesicles but not to distal sites for processing. Anti-GFP or anti-DsRed immunoblots of cell fractions from A549 cells nontransfected (lane a) or transfected with EGFPC1 alone (top panel), EGFP/SP-C^WT (second panel from top), DsRed/Chimera (third panel from top), and EGFP/SP- C^{SPDDY→LLLWKN} (bottom panel). Blots of whole cell lysates (lanes a, b), postcentrifuge supernatant prior (lane e) and following (lane f) sodium carbonate treatment of initial pellet (lane b), and postcentrifuge pellet (lane d) products are shown. The primary product of EGFP protein alone migrates to 27 kDa, while the primary products of EGFP/SP-C^WT proprotein and EGFP/SP- C^{SPDDY→LLLWKN} migrate to 48 kDa, and DsRed/Chimera fusion protein migrates to 47 kDa. Processed product of EGFP/SP-C^WT (arrowhead) is abundant, whereas neither DsRed/Chimera nor EGFP/SP-C^{SPDDY→LLLWKN} is adequately processed (arrowheads) (lanes b, d, e). *Cleaved peptide artifact from the EGFP vector.

Alanine mutation of a LLLWKN results in ER retention. Representative fluorescence microscopy of A549 cells expressing hABCA3/DsRed or hABCA3/EGFP (A, B, C) and mABCA1/FLAG (D, E, F) mutants, where the entire LLLWKN motif (B, E), residues 3-8 (A), 4-8 (D), 15-19 (C), or 15-17 (F) were substituted with alanine. Cells were fixed for subsequent direct fluorescence visualization or stained for the ER marker calnexin and a Texas Red or FITC secondary antiserum. Vesicular colocalization (A, C, D, F) and noncolocalization (B, E, left panels) of EGFP or DsRed SP-C plasmids that were concomitantly transfected with the mutant transporters are shown. mABCA1/FLAG mutants were immunostained with either Texas Red- (D, E-left panel, F) or FITC- (E, right panel) tagged antibodies. Colocalizations of Texas Red- and FITC-tagged calnexin with the mutants are also shown (B, E, right panels). Regions outlined by the solid boxes were enlarged to provide additional resolution (bottom panels). Bar, 5 µm.

Wild-type mABCA1 and hABCA3 are trafficked to post-Golgi vesicles. The subcellular distribution of wild-type hABCA3/EGFP (A) and mABCA1/FLAG (B) constructs was determined by fluorescence microscopy. Plasmid cDNAs encoding the constructs were introduced, both alone (A, B, top rows) or with SP-C proprotein (A, B, bottom rows), into A549 cells grown on coverslips. At 16-24 h following transfection, cells were fixed for subsequent fluorescence visualization or stained for the lysosomal-like organelle marker CD63 and a Texas Red secondary antibody. The mABCA1/FLAG was stained for FLAG (using anti-FLAG antibody) conjugated with either FITC (B, top row) or Texas Red (B, bottom row) secondary antiserum. (C) hABCA3 and mABCA1 were transfected at a 1:1 concentration and visualized by confocal microscopy. Regions outlined by the solid boxes were enlarged to provide additional resolution (right panels). Results are from at least three separate experiments. Bar, 5 µm. (D) % vesicle colocalization of expressed hABCA3^WT/DsRed or mABCA1/FLAG proteins with EGFP/SP-C (A, B, bottom rows). At least 100 cells expressing both proteins and at least 150 vesicles per cell from three separate experiments were counted. *P < 0.01 compared with hABCA3.

The LLLWKN motif can target an unrelated protein to post-Golgi vesicles. (A, top row) Representative confocal images of A549 cells of a DsRed-tagged chimeric proSP-C construct made by swapping the entire N-terminal (35 amino acid) domain of proSP-C with the N-terminal (21 amino acid) domain of hABCA3. The DsRed/Chimera shows partial colocalization in vesicles containing hABCA3^WT/EGFP. (A, middle row) Only the targeting motif of proSP-C (SPPDY) was substituted with LLLWKN. Partial colocalization of the DsRed/SP-C^{SPDDY→LLLWKN} with hABCA3^WT/EGFP is shown. (A, bottom row) EGFP/SP-C^{SPDDY→LLLWKN} expressing A549 cells show partial colocalization in compartments positive for the lysosomal-like vesicle marker CD63. Regions outlined by solid boxes were enlarged to provide additional resolution (right panels). Bar, 5 µm. (B) % vesicle colocalization count between expressed hABCA3^WT and SP-C^WT (, bottom row), chimera (A, top row), or SP-C^{SPDDY→LLLWKN} (A, middle row). At least 100 cells expressing both proteins and at least 150 vesicles per cell from three separate experiments were counted. *P < 0.01 compared with DsRed/SP-C^WT. (C) Representative confocal images of A549 cells expressing EGFP/SP-C^WT and DsRed/SP-C^{SPDDY→LLLWKN} in the absence (left columns) and presence (right columns) of monensin. Merged images show colocalization of the SP-C isoforms in compartments positive for the trans-Golgi marker P230 (right columns). Regions delineated by the solid boxes were enlarged to provide additional resolution (bottom rows). Bar, 5 µm.

Scanning alanine mutagenesis of the LLLWKN motif reveals distinct subcellular distribution patterns. The subcellular distribution of various alanine mutant constructs for the hABCA3 LLLWKN motif was determined by fluorescence microscopy. Plasmid DNAs encoding a range of alanine mutant constructs of the hABCA3 LLLWKN motif together with the SP-C proprotein construct were introduced into A549 cells. (A) 16-24 h following transfection, cells were fixed for fluorescence imaging. For those mutants that displayed reticular expression, cells were stained for calnexin and a Texas Red secondary antibody. (Panels a–e) Group I mutants that included hABCA3^ALLWKN/EGFP, hABCA3^LLAWKN/EGFP, hABCA3^ALAWKN/EGFP, hABCA3^LLLWKA/EGFP, and hABCA^ALLWKA/EGFP revealed vesicular distribution predominantly in SP-C-positive vesicles. (Panels f–l) Group II mutants that included hABCA3^LALWKN/EGFP, hABCA3^AALLWKN/EGFP, hABCA3^LAAWKN/EGFP, hABCA3^LLLWAN/EGFP, hABCA3^LLLWAA/EGFP, hABCA3^AAAWKN/EGFP, and hABCA3^LALWAA/EGFP displayed both vesicular and reticular distributions that were positive for SP-C and calnexin, respectively. (Panels m, n) Group III mutants, hABCA3^LAAAAN/EGFP and hABCA3^LAAAAA/EGFP, were only expressed in calnexin-positive compartments. Regions delineated by the solid boxes were enlarged to provide additional resolution (right panels). Bar, 5 µm. (B) Quantitative cell expression patterns for hABCA3/EGFP mutant proteins. Colocalization of DsRed/SP-C or calnexin marker with each hABCA3/EGFP mutant was determined by manual counting 16-24 h following transfection. At least 150 cells expressing both proteins or protein and marker from three separate experiments were evaluated. *P < 0.001, **P < 0.05 compared with wild-type hABCA3.