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Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Rat surfactant protein (SP) C is synthesized as a 194-amino acid proprotein that is proteolytically processed to a 35-amino acid mature form in subcellular compartments distal to the medial Golgi compartment. To identify domains of SP-C proprotein (proSP-C) necessary for endoplasmic reticulum translocation and for targeting to cytosolic processing compartments, we characterized expression patterns of heterologous SP-C fusion proteins in A549 lung epithelial cells and in the rat pheochromocytoma cell line PC-12. cDNA constructs were produced; these constructs encoded fusion proteins consisting of enhanced green fluorescent protein (EGFP) and wild-type proSP-C (EGFP/SP-C1-194), mature SP-C (EGFP/SP-C24-59), or progressive deletions of the NH2- or COOH-terminal flanking domains. By fluorescence microscopy, EGFP/SP-C1-194 transfected into A549 cells was translocated and expressed in acidic cytoplasmic vesicles. By deletional analysis, a functional signal peptide was mapped to the domain Phe24 to His59, whereas a motif for targeting to cytosolic vesicular compartments was localized to the NH2 flanking domain Met10 to Gln23. Truncations of the distal COOH terminus were retained in the endoplasmic reticulum/Golgi compartment; however, the COOH flanking region alone was insufficient for targeting. In PC-12 cells, EGFP/SP-C1-194 was expressed in peripheral cytosolic vesicles, whereas EGFP/SP-C24-194 and EGFP/SP-C24-59 were each translocated but not targeted. We conclude that two domains in the proSP-C sequence are required for targeting: mature SP-C (Phe24 to Leu58) contains a functional signal sequence active in epithelial and nonepithelial cells, whereas Met10 to Gln23, but not the COOH flanking peptide, is required for targeting to late vesicular compartments.
surfactant protein C; propeptide targeting; A549 cells; green fluorescent protein
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