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 NH2-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 NH2 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/SPC-(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 cotransfected with pcDNA3/SP-C-(1–194), both EGFP/SPC-(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, 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/SPC-(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. Surfactant protein C (SP-C), isolated from bronchoalveolar lavage (BAL) of a variety of mammalian species represents one of the most hydrophobic secretory proteins characterized (1–3). The alveolar form of SP-C (mature SP-C) is a 35-amino acid peptide (Mr 3,500) that contains an extremely high content of branched aliphatic side-chains as well as covalently attached palmitic acid at two adjacent cysteine residues in its NH2 terminus (2). Mature SP-C partitions with surfactant phospholipid in organic extracts of BAL, and both purified natural or recombinant forms impart properties of rapid surface absorption and low surface tension to reconstituted synthetic lipids (4). In a lipid environment, the secondary structure of mature SP-C is predominantly -helical (5) and exhibits an extended lipophilic surface with positive charges located near the NH2 terminus. The valyl-rich monomeric -helical state transforms into insoluble -sheet aggregates upon incubation in aqueous solvents (6). A dimeric form of SP-C has been recovered from BAL of several species, although the functional significance of this variant is unknown (7). Synthesized exclusively by the alveolar type 2 cell as a 21kDa proprotein precursor (proSP-C21), the mature SP-C domain is positioned asymmetrically within the primary translation product flanked by propeptides of 23 amino acids at the NH2 terminus and 136 residues at the COOH terminus (1–3, 8). Production of SP-C from this larger precursor is a multistep process requiring four discrete proteolytic cleavages, all occurring in subcellular compartments located distal to the medialGolgi (9–14). Thus, translocation of synthesized proSP-C21 to the ER and targeting of proprotein from the Golgi to distal vesicular organelles (i.e. multivesicular bodies and lamellar bodies) are crucial for biogenesis and secretion of the mature alveolar form. Several important structural domains that influence proSP-C trafficking have been defined (9, 15). By deletion analyses, the mature SP-C domain (Phe–Leu) acting as a functional signal-anchor sequence was shown to mediate ER translocation (9). In vitro, the primary translation product has been shown to be a bitopic, transmembrane protein, anchored by the mature SP-C region in a type II (NH2, cytosol/COOH, lumen) orientation (15, 16). ProSP-C remains in an integral membrane form during trafficking from the ER to the lamellar body, while undergoing proteolytic cleavage of the extramembrane propeptide regions (9, 13). Using both in vitro and in vivo models, the intracellular targeting motif has been localized to the cytosolic (NH2-terminal) portion of proSP-C (9, 15). Fusion proteins containing EGFP and proSP-C mutants lacking conserved amino * This work was supported by National Institutes of Health Grants HL-19737 and P50-HL56401 (both to M. F. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, 807 BRB II/III Bldg. , 421 Curie Blvd., Philadelphia, PA 19104-6160. Fax: 215-573-4469; E-mail: mfbeers@mail.med.upenn.edu. 1 The abbreviations used are: SP-C, pulmonary surfactant protein C (3.7 kDa); BAL, bronchoalveolar lavage; BMH, bismaleimidohexane; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HA, hemagglutinin antigen; PBS, phosphate-buffered saline; TR, Texas red. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 22, Issue of May 31, pp. 19929–19937, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.