Hinners, Ina; (2003) Molecular mechanisms of clathrin coat formation in neuroendocrine cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Neuroendocrine cells possess specialised organelles, secretory granules, that comprise the storage compartment for bioactive molecules, such as hormones. These granules fuse with the plasma membrane upon a specific signal to release their contents into the extracellular space. They undergo a maturation process that involves protein modification and sorting, membrane remodelling and acidification of the granule. Immature secretory granules (ISGs) contain a clathrin coat that is not found on mature secretory granules. This coat may be involved in the protein- and membrane-remodelling process occuring during granule maturation. I have investigated the mechanism that leads to clathrin coat formation on ISGs. The small GTPase ARF (ADP ribosylation factor) is necessary for the recruitment of the clathrin adaptor complex-1 (AP-1) to ISGs. I have also found that ARF interacts with AP- 1. In vitro recruitment of ARF can be reconstituted using isolated ISGs, purified myristoylated ARF1, and the non-hydrolysable GTP analogue GTPγS. Recruitment of ARF is inhibited by treatment of ISGs with trypsin and by the fungal metabolite Brefeldin A (BFA). These data suggest ISGs contain a BFA-sensitive exchange factor for ARF. However, this factor remains to be identified. The mechanism that links coat formation and cargo selection during vesicle biogenesis remains elusive to date. It has been hypothesised that SNARE proteins can recruit coat proteins, i.e. initiate vesicle biogenesis. To test this hypothesis, I have investigated how the SNARE proteins VAMP4 and syntaxin 6 are directed into clathrin coated vesicles. VAMP4 contains a di-leucine motif that confers binding to AP-1. In addition, VAMP4 is a substrate for the protein kinase CK2. I have shown that serine 30 is the phosphorylation site, and that phosphorylation of VAMP4 enhances the interaction with AP-1. VAMP4 is also found in a complex with PACS-1 in vivo, and my data would suggest that the enhanced interaction of phosphorylated VAMP4 and AP-1 may be mediated by PACS-1. I could not detect a direct interaction between syntaxin 6 and clathrin adaptor complexes. Although VAMP4 and syntaxin 6 are efficiently sorted into clathrin coated vesicles, I have found no experimental evidence to support the hypothesis, that these molecules might inititate clathrin coated vesicle formation.

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