Biosynthesis of surfactant protein C (SP-C). Sorting of SP-C proprotein involves homomeric association via a signal anchor domain.
Rat surfactant protein C (SP-C) is synthesized as a 194-amino acid propeptide (SP-C-(1-194)) that is directed to the distal secretory pathway and proteolytically processed as an integral membrane protein to yield its mature form. We had shown previously that trafficking of proSP-C is mediated both by a signal anchor domain contained within the mature SP-C sequence and by a targeting domain in the NH(2)-flanking propeptide. Based on evidence from other integral membrane proteins, we hypothesized that proSP-C targeting is effected by oligomerization of proSP-C monomers. To evaluate this in vitro, cDNA constructs encoding for either wild type proSP-C (pcDNA3/SP-C-(1-194)) or heterologous fusion proteins containing green fluorescent protein (EGFP) linked to SP-C-(1-194) (EGFP/SP-C-(1-194)), to mutant proSP-C lacking the NH(2) targeting domain (EGFP/SP-C-(24-194)), or to mature SP-C alone (EGFP/SP-C-(24-58)) were produced. In transfected A549 cells, fluorescence microscopy revealed that pcDNA3/SP-C-(1-194) and EGFP/SP-C-(1-194) were each expressed in CD63 (+), EEA1 (-) cytoplasmic vesicles. Expression of EGFP/SP-C-(24-194) or EGFP/SP-C-(24-58) resulted in translocation but retention in early compartments. When co-transfected with pcDNA3/SP-C-(1-194), both EGFP/SP-C-(24-194) and EGFP/SP-C-(24-58) were directed to CD63 (+) vesicles that also contained SP-C-(1-194). In contrast, trafficking of a folding mutant that forms juxtanuclear aggregates, EGFP/SP-C(C122/186G), was not corrected by cotransfection with pcDNA3/SP-C-(1-194). Chemical cross-linking studies of transfected cell lysates with bismaleimidohexane produced multimeric forms of both EGFP/SP-C-(1-194) and EGFP/SP-C-(24-58). These results indicate that sorting involves oligomeric association of proSP-C monomers mediated by the mature SP-C domain. Heteromeric assembly allows wild type proSP-C to facilitate trafficking of SP-C mutants with intact transmembrane domains but lacking targeting signals. We speculate that heterotypic oligomerization of wild type with SP-C folding mutants produces a dominant negative thus contributing to the pathology of chronic lung disease associated with patients heterozygous for mutant SP-C alleles.
Wang
Wen-Jing
WJ
Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Russo
Scott J
SJ
Mulugeta
Surafel
S
Beers
Michael F
MF
D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006579:Heterozygote; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000096:biosynthesis* / Q000737:chemistry*; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011993:Recombinant Fusion Proteins; D003545:Cysteine