Abstract
Biosynthesis of surfactant protein C (SP-C) by alveolar type 2 cells requires proteolytic processing of a 21-kDa propeptide (proSP-C21) in post-Golgi compartments to yield a 3.7-kDa mature form. Scanning alanine mutagenesis, binding assays, and co-immunoprecipitation were used to characterize the proSP-C targeting domain. Delivery of proSP-C21 to distal processing organelles is dependent upon the NH2-terminal cytoplasmic SP-C propeptide, which contains a conserved PPDY motif. In A549 cells, transfection of EGFP/proSP-C21 constructs containing polyalanine substitution for Glu11-Thr18, 13PPDY16, or 14P,16Y produced endoplasmic reticulum retention of the fusion proteins. Protein-protein interactions of proSP-C with known WW domains were screened using a solid-phase array that revealed binding of the proSP-C NH2 terminus to several WW domains found in the Nedd4 family of E3 ligases. Specificity of the interaction was confirmed by co-immunoprecipitation of proSP-C and Nedd4 or Nedd4-2 in epithelial cell lines. By Western blotting and reverse transcription-PCR, both forms were detected in primary human type 2 cells. Knockdown of Nedd4-2 by small interference RNA transfection of cultured human type 2 cells blocked processing of 35S-labeled proSP-C21. Mutagenesis of potential acceptor sites for ubiquitination in the cytosolic domain of proSP-C (Lys6, Lys34, or both) failed to inhibit trafficking of EGFP/proSP-C21. These results indicate that PPDY-mediated interaction with Nedd4 E3-ligases is required for trafficking of proSP-C. We speculate that the Nedd4/proSP-C tandem is part of a larger protein complex containing a ubiquitinated component that further directs its transport.
Highlights
Using deletional mutagenesis, we had previously shown that the NH2-terminal propeptide flanking domain (NTP) of proSP-C, which is oriented into the cytosol, contained potential targeting motifs for post-Golgi trafficking [8, 9]
Mutation of the NH2-flanking Propeptide to Remove a PPDY Motif Blocks Transport and Processing of proSP-C—To characterize the role of NH2-flanking propeptide in surfactant protein C (SP-C) trafficking, EGFP fusion proteins containing polyalanine substitutions of the proSP-C NH2 terminus were evaluated after transfection into A549 cells
Within 48 h of introduction of plasmid cDNA, expression of the EGFP/SP-C1–194 fusion protein was detected in cytoplasmic vesicles (Fig. 2), which co-localizes with CD63, a marker of lysosomal-like organelles [24, 25]
Summary
The pEGFP-C1 plasmid was purchased from Clontech. Tissue culture medium was produced by the Cell Center Facility, University of Pennsylvania. Horseradish peroxidase-conjugated secondary antibodies were obtained from Bio-Rad. EGFP/Nedd (⌬C2 clone) and EGFP/Nedd plasmids, previously characterized [17, 23], were the generous gift of Dr Peter Snyder at the University of Iowa. A chimera consisting of EGFP fused to the NH2 terminus of wild-type rat SP-C (EGFP/SP-C1–194) was cloned into the EGFPC1 expression vector and has been previously characterized [9]. Using this as template, EGFP-mutant SP-C chimeras containing an NH2-terminal truncation (SP-C19–194) or internal deletion (SP-C⌬10–18) were subsequently produced and characterized [8].
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