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.
Bibliographical noteFunding Information:
We thank Kamburpola Jayawardena and Yagnesh Umrania for help with the proteomics. We also thank the UMN Imaging Center at the University of Minnesota. B.S.S. acknowledges the members of the Robinson, Bartolomucci, and Mahata laboratories. 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.
© 2017 Sahu et al.