title: Biosynthesis of surfactant protein C: characterization of aggresome formation by EGFP chimeras containing propeptide mutants lacking conserved cysteine residues.
abstract: Surfactant protein C (SP-C) is a lung-specific secreted protein, which is synthesized as a 21-kDa propeptide (SP-C(21)) and then proteolytically processed as a bitopic transmembrane protein in subcellular compartments distal to the medial Golgi to produce a 3.7 kDa mature form. We have shown that initial processing of SP-C(21) involves two endoproteolytic cleavages of the C terminus and that truncation of nine amino acids from the C-flanking peptide resulted in retention of mutant protein in proximal compartments. Because these truncations involved removal of a conserved cysteine residue (Cys(186)), we hypothesized that intralumenal disulfide-mediated folding of the C terminus of SP-C(21) is required for intracellular trafficking. To test this, cDNA constructs encoding heterologous fusion proteins consisting of enhanced green fluorescent protein (EGFP) attached to the N terminus of wild-type rat proSP-C (EGFP/SP-C(1-194)), C-terminally deleted proSP-C (EGFP/SP-C(1-185); EGFP/SP-C(1-191)) or point mutations of conserved cysteine residues (EGFP/SP-C(C122G); EGFP/SP-C(C186G); or EGFP/SP-C(C122/186G)) were transfected into A549 cells. Fluorescence microscopy revealed that transfected EGFP/SP-C(1-194) and EGFP/SP-C(1-191 )were expressed in a punctate pattern within CD-63 positive, EEA-1 negative cytoplasmic vesicles. In contrast, EGFP/SP-C(1-185), EGFP/SP-C(C122G), EGFP/SP-C(C186G) and EGFP/SP-C(C122/186G) were expressed but retained in a juxtanuclear compartment that stained for ubiquitin and that contained (&ggr;)-tubulin and vimentin, consistent with expression in aggresomes. Treatment of cells transfected with mutant proSP-C with the proteasome inhibitor lactacysteine enhanced aggresome formation, which could be blocked by coincubation with nocodazole. Western blots using a GFP antibody detected a single form in lysates of cells transfected with EGFP/SP-C cysteine mutants, without evidence of smaller degradation fragments. We conclude that residues Cys(122) and Cys(186) of proSP-C are required for proper post-translational trafficking. Mutation or deletion of one or both of these residues results in misfolding with mistargeting of unprocessed mutant protein, leading to formation of stable aggregates within aggresomes.
authors.affiliation: Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: A F
authors.initials: AF
authors.lastname: Kabore
authors.forename: W J
authors.initials: WJ
authors.lastname: Wang
authors.forename: S J
authors.initials: SJ
authors.lastname: Russo
authors.forename: M F
authors.initials: MF
authors.lastname: Beers
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D017124:Conserved Sequence; D003545:Cysteine; D003546:Cysteine Endopeptidases / Q000378:metabolism; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008168:Lung; D009097:Multienzyme Complexes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D011510:Proteolipids / Q000096:biosynthesis* / Q000235:genetics*; D037721:Pulmonary Surfactant-Associated Protein C; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020545:Respiratory Mucosa / Q000166:cytology / Q000502:physiology / Q000648:ultrastructure; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D017931:DNA Primers; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D010455:Peptides; D011510:Proteolipids; D037721:Pulmonary Surfactant-Associated Protein C; D011663:Pulmonary Surfactants; D011993:Recombinant Fusion Proteins; C489679:SFTPC protein, human; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D003545:Cysteine