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Anterograde transport of surfactant protein C proprotein to distal processing compartments requires PPDY-mediated association with Nedd4 ubiquitin ligases.

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.

Pubmed ID: 19366705

Authors

  • Kotorashvili A
  • Russo SJ
  • Mulugeta S
  • Guttentag S
  • Beers MF

Journal

The Journal of biological chemistry

Publication Data

June 12, 2009

Associated Grants

  • Agency: NHLBI NIH HHS, Id: HL-059959
  • Agency: NHLBI NIH HHS, Id: HL-19737
  • Agency: NHLBI NIH HHS, Id: HL090732
  • Agency: NHLBI NIH HHS, Id: R01 HL059959

Mesh Terms

  • Cells, Cultured
  • Endosomal Sorting Complexes Required for Transport
  • Epithelial Cells
  • Green Fluorescent Proteins
  • Humans
  • Lung
  • Mutagenesis
  • Mutant Chimeric Proteins
  • Protein Precursors
  • Protein Transport
  • Pulmonary Surfactant-Associated Protein C
  • RNA, Small Interfering
  • Transfection
  • Ubiquitin-Protein Ligases
  • Ubiquitination