Structural requirements for intracellular targeting of SP-C proprotein.
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-C(1-194)), mature SP-C (EGFP/SP-C(24-59)), or progressive deletions of the NH(2)- or COOH-terminal flanking domains. By fluorescence microscopy, EGFP/SP-C(1-194) transfected into A549 cells was translocated and expressed in acidic cytoplasmic vesicles. By deletional analysis, a functional signal peptide was mapped to the domain Phe(24) to His(59), whereas a motif for targeting to cytosolic vesicular compartments was localized to the NH(2) flanking domain Met(10) to Gln(23). 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-C(1-194) was expressed in peripheral cytosolic vesicles, whereas EGFP/SP-C(24-194) and EGFP/SP-C(24-59) were each translocated but not targeted. We conclude that two domains in the proSP-C sequence are required for targeting: mature SP-C (Phe(24) to Leu(58)) contains a functional signal sequence active in epithelial and nonepithelial cells, whereas Met(10) to Gln(23), but not the COOH flanking peptide, is required for targeting to late vesicular compartments.
Russo
S J
SJ
Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Wang
W
W
Lomax
C A
CA
Beers
M F
MF
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D017931:DNA Primers; D066246:ErbB Receptors / Q000235:genetics; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000166:cytology* / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism; D016716:PC12 Cells; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D066246:ErbB Receptors