Post-translational processing of surfactant protein-C proprotein -: Targeting motifs in the NH2-terminal flanking domain are cleaved in late compartments

Rat surfactant protein (SP)-C is a 3,7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C-21). We have demonstrated that initial post-translational processing of SP-C-21 involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20318-20328). The goal of the current study was to define processing and function of the NH2-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH2 terminus, NPROSP-C2-9 (epitope = D-2-L-9) and NPROSP-C11-23 (= E-11-Q(23)) were produced. By Western blotting, both antisera identified SP-C-21 in microsomes. A 6-kD form (SP-C-6), enriched in lamellar bodies (LBs), was detected only by NPROSP-C11-23 and not extractable with NaCO3 treatment. Immunogold staining of ultrathin lung sections with NPROSP-C11-23 identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C2-9 labeled only mvb. S-35-pulse chase analysis demonstrated synthesis of SP-C-21 and three intermediate forms (SP-C-16 SP-C-7 and SP-C-6). Complete processing involved four separate cleavages with a precursor-product relationship between the low molecular weight forms SP-C-7 and SP-C-6, Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH2-terminaI deletion mutants showed targeting of EGFP/SP-C1-194 and EGFP/SP-C10-194 to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C19-194 EGFP/SP-CDelta 10-18, and EGFP/SP-C24-194 were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH2 terminus (M-1-L-9), which occurs after removal of COOH-flanking domains (H-59-I-194) but before packaging in LBs, and that the region M-10-T-18 is required for targeting of proSP-C to post-ER vesicular compartments in the biosynthetic pathway.