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Role of clathrin in dense core vesicle biogenesis
- Source :
- Molecular Biology of the Cell
- Publication Year :
- 2016
-
Abstract
- Knocking down clathrin in PC12 cells not only affects the maturation of dense core vesicles, it also renders them essentially incapable of secretagogue-induced exocytosis.<br />The dense core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible short hairpin RNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were approximately fourfold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced approximately twofold in clathrin-depleted cells as a whole and approximately fivefold in vesicle-enriched fractions. Our combined data sets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle.
- Subjects :
- 0301 basic medicine
Cell
Biology
Clathrin
PC12 Cells
Exocytosis
Mass Spectrometry
Small hairpin RNA
03 medical and health sciences
Neuroendocrine Cells
medicine
Animals
Secretion
Molecular Biology
Cells, Cultured
Vesicle
Secretory Vesicles
Calcium-Binding Proteins
Cell Biology
Articles
Cell biology
Rats
030104 developmental biology
medicine.anatomical_structure
Membrane Trafficking
Clathrin Heavy Chains
biology.protein
Cell fractionation
Biogenesis
Subcellular Fractions
Subjects
Details
- ISSN :
- 19394586
- Volume :
- 28
- Issue :
- 20
- Database :
- OpenAIRE
- Journal :
- Molecular biology of the cell
- Accession number :
- edsair.doi.dedup.....a72e04ab831e687801abdd692eef58fe