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Abstract: SA-OR054

Proteomic Analysis of Clathrin-Coated Vesicles from Podocytes Identifies Cargo Proteins

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Groener, Marwin, University Clinic Erlangen, Erlangen, Germany
  • Inoue, Kazunori, Osaka University Graduate School of Medicine, Suita Osaka, Japan
  • Cross, Elizabeth, Yale University School of Medicine, New Haven, Connecticut, United States
  • Ishibe, Shuta, Yale University School of Medicine, New Haven, Connecticut, United States
Background

Clathrin-mediated endocytosis (CME) plays a crucial role in podocyte health. Knockout of proteins involved in CME resulted in severe albuminuria and foot process effacement in mice. However, the cargo of clathrin-coated vesicles (CCVs) in podocytes is unknown. The goal of this study was to isolate CCVs from podocytes and identify their cargo by proteomic analysis.

Methods

Kidneys were isolated from Podocin-Cre Rosa-DTRflox mice. The glomeruli were seeded and treated with diphtheria toxin to obtain pure primary podocyte cultures. After cell harvesting, CCVs were isolated by D2O-sucrose density gradient centrifugation using multiple ultracentrifugation steps. Enrichment of CCVs was assessed by immunoblotting and electron microscopy (EM). LC-mass spectrometry (LC-MS) was performed for proteomic analysis. Proteins with higher abundance than transferrin receptor protein 1 were evaluated for CCV cargo potential by comparison to published impurities in CCV preparations, podocyte proteomic databases, and by searching the literature for CME-association.

Results

Immunoblotting for multiple protein markers of CME revealed enrichment in the CCV fraction. Enrichment of CCVs amongst other small vesicles was observed on electron microscopy. Clathrin-heavy chain was the fourth-most abundant protein in LC-MS analysis of the vesicle fraction. Proteomics yielded a total of over 1700 proteins. After adjustment for impurities and upregulation from whole cell expression, over 50 potential cargo proteins were identified. Among those are fibronectin, receptor of activated protein C kinase, thrombospondin-1, and vigilin.

Conclusion

This is the first time CCVs were enriched from podocytes. Enrichment was confirmed by immunoblotting, EM and LC-MS analysis. Proteomic analysis of CCV cargo and adjustment for impurities identified the most abundant cargo proteins in podocytes. These findings help to elucidate the importance of endocytic trafficking for podocyte health and disease.

Funding

  • NIDDK Support