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Role of clathrin in dense core vesicle biogenesis

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Sahu, BS 
Manna, PT 
Edgar, JR 
Antrobus, R 
Mahata, SK 


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 shRNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were ∼4-fold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced ∼2-fold in clathrin-depleted cells as a whole and ∼5-fold in vesicle-enriched fractions. Our combined datasets 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.



Animals, Calcium-Binding Proteins, Cells, Cultured, Clathrin, Clathrin Heavy Chains, Exocytosis, Mass Spectrometry, Neuroendocrine Cells, PC12 Cells, Rats, Secretory Vesicles, Subcellular Fractions

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Molecular Biology of the Cell

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American Society for Cell Biology
Wellcome Trust (086598/Z/08/Z)
Wellcome Trust (100140/Z/12/Z)
Wellcome Trust (093026/Z/10/Z)
This work was funded by grants from the Wellcome Trust: 086598 (to M.S.R.), 100140 (Wellcome Trust Strategic Award), and 093026 (for the FEI Tecnai G2 Spirit BioTWIN transmission EM); and by a National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases grant (R01DK102496) to A.B.