Sorting of Cargo Proteins Within the Regulated Secretory Pathway: the Peripheral Membrane Protein HID-1 as a Sorting and Vesicle Biogenesis Factor

<p>Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize the TGN peripheral membrane protein, HID-1, as a LDCV biogenesis factor. We show that ablation of HID-1 results in defects in cargo storage and regulated secretion, a reduction in the number of LDCVs with perturbed morphology and biochemical properties, and results in defects in TGN acidification. Despite the importance of HID-1 to regulated secretion, little is known about HID-1 domain architecture and it is unclear how HID-1 associates to the TGN. We report that the N-terminus of HID-1 mediates membrane binding through a myristoyl group in combination with an unidentified binding motif within the C-terminus, and an interaction with the Golgi protein, Golga5, allows for targeting to the TGN. Finally, we identify the C-terminus as the functional domain of HID-1, capable of rescuing the secretion and sorting defects. A point mutation within that domain, identified in patients with endocrine and neurological deficits, is not functional. We propose that HID-1 N-terminal and C-terminal domains, in cooperation with Golga5, ensure specific binding to Golgi membranes and functionality of HID-1.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Year dc:date.available

2020

Author dc:creator

• Hummer, Blake H.

Contributors dc:contributor

• Cedric S. Asensio
• Joseph Angleson
• J. Todd Blankenship
• Dinah Loerke
• Schuyler Van Engelenburg

• HID-1
• Peripheral membrane protein
• Regulated secretion
• Biochemistry
• Biochemistry, Biophysics, and Structural Biology
• Cell and Developmental Biology
• Cell Biology
• Life Sciences
• Molecular Biology

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/1781

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-2777