Role of clathrin in dense core vesicle biogenesis
View / Open Files
Authors
Sahu, BS
Antrobus, R
Mahata, SK
Bartolomucci, A
Borner, GHH
Publication Date
2017-10-01Journal Title
Molecular Biology of the Cell
ISSN
1059-1524
Publisher
American Society for Cell Biology
Volume
28
Issue
20
Pages
2676-2685
Language
eng
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Sahu, B., Manna, P., Edgar, J., Antrobus, R., Mahata, S., Bartolomucci, A., Borner, G., & et al. (2017). Role of clathrin in dense core vesicle biogenesis. Molecular Biology of the Cell, 28 (20), 2676-2685. https://doi.org/10.1091/mbc.E16-10-0742
Abstract
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.
Sponsorship
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.
Funder references
Wellcome Trust (086598/Z/08/Z)
Wellcome Trust (100140/Z/12/Z)
Wellcome Trust (093026/Z/10/Z)
Identifiers
External DOI: https://doi.org/10.1091/mbc.E16-10-0742
This record's URL: https://www.repository.cam.ac.uk/handle/1810/267753
Rights
Attribution-NonCommercial-ShareAlike 4.0 International, Attribution-NonCommercial-ShareAlike 4.0 International, Attribution-NonCommercial-ShareAlike 4.0 International, Attribution-NonCommercial-ShareAlike 4.0 International
Statistics
Total file downloads (since January 2020). For more information on metrics see the
IRUS guide.
Recommended or similar items
The following licence files are associated with this item: