Structural requirements for intracellular targeting of SP-C proprotein

Abstract

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.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

“…Plasmids consisting of enhanced green fl uorescent protein (EGFP) and DsRed fused to the N terminus of wild-type human SP-C (EGFP/SP-C ) were cloned into the EGFPC1 and DsRedC1 expression vectors and have been previously characterized ( 17,23 ). N-terminus-tagged (instead of C-terminus-tagged) SP-C isoforms were chosen because the SP-C proprotein is cleaved in the distal C terminus (at residue ‫ف‬ 145) in a non-cell-specifi c manner at the early stage of post-Golgi traffi cking ( 24,25 ) ( Fig.…”

“…Moreover, DsRed ABCA3/SP-C chimera containing the N-terminal domain of ABCA3 (ABCA3 ) and C-terminal domain of proSP-C (SP-C ) were produced. For these constructs, a single round, two primers (include restriction sites) PCR amplifi cation and subcloning strategy was used as described previously ( 17 ) with the oligonucleotide sets in supplementary Table I.…”

“…In addition to large domain signals with complex structural architecture, small-size motifs containing short spanning amino acid residues have been shown to serve as major determinants in directing the intracellular routes and localization of protein to their site of function or secretion. Synaptic vesicle-targeted proteins ( 15 ), insulin receptor sorting to lysosomes ( 16 ), surfactant protein C routing to the lamellar body of alveolar type II cells (17)(18)(19), and the adherens junction-targeted shrew-1 transmembrane proteins ( 20 ) all represent proteins containing small targeting motifs that play a role in guiding proteins through proper intracellular routes and/or to their fi nal destinations.…”

Biosynthesis of Surfactant Protein C (SP-C)

“…Cell Transfection-A549 cells, grown to 50% confluence on glass coverslips in 35-mm plastic dishes, were transiently transfected with single cDNA constructs (10 g/dish) or co-transfected with two different constructs (5 g/construct/dish) by CaPO 4 precipitation as previously described (9,10,18). The medium was replaced at 24 h, and transfected cells were maintained for up to 48 h.…”

“…Using both in vitro and in vivo models, the intracellular targeting motif has been localized to the cytosolic (NH 2 -terminal) portion of proSP-C (9,15). Fusion proteins containing EGFP and proSP-C mutants lacking conserved amino acid residues in the NH 2 domain (Glu 11 -Thr 19 ) are retained in calnexin-positive (ER) compartments (9,10). Intratracheal injection of mice with adenovirus constructs encoding hemagglutinin (HA)-tagged SP-C proteins has confirmed that the NH 2 domain (Phe 1 -Glu 23 ), in conjunction with the mature SP-C domain, is sufficient for secretion of SP-C (15).…”