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.
Kotorashvili
Adam
A
From the Surfactant Biology Laboratories, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104.
Russo
Scott J
SJ
From the Surfactant Biology Laboratories, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104.
Mulugeta
Surafel
S
From the Surfactant Biology Laboratories, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104.
Guttentag
Susan
S
Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104.
Beers
Michael F
MF
From the Surfactant Biology Laboratories, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104. Electronic address: mfbeers@mail.med.upenn.edu.
D002478:Cells, Cultured; D056827:Endosomal Sorting Complexes Required for Transport; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008168:Lung / Q000166:cytology; D016296:Mutagenesis; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D037721:Pulmonary Surfactant-Associated Protein C / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D056827:Endosomal Sorting Complexes Required for Transport; D050506:Mutant Chimeric Proteins; D011498:Protein Precursors; D037721:Pulmonary Surfactant-Associated Protein C; D034741:RNA, Small Interfering; C489679:SFTPC protein, human; D049452:Green Fluorescent Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases