This application addresses several important issues for understanding the role of Munc13 proteins as tethering/priming factors for membrane fusion. At multiple stations in the secretory and endosomal trafficking pathways, vesicle-target membrane interactions are mediated by a diverse group of tethering factors. These factors recognize vesicle components and target membrane constituents to achieve vectorial targeting. Tethering factors also actively engage SNARE proteins to assemble trans SNARE complexes for fusion. In the regulated secretory pathway of neuroendocrine cells, vesicle priming involves SNARE complex assembly and requires Munc13 family proteins. Munc13 family proteins have sequence similarity in SNARE-binding domains that are homologous to tethering factors. We found that three members of the Munc13 protein family (Munc13- 1, Munc13-4, CAPS) exhibit similar SNARE-binding properties and similarly promote SNARE complex formation and SNARE-dependent liposome fusion. Surprisingly, ubMunc13-2 and CAPS-1 functioned non- redundantly and cooperatively in evoked DCV (dense-core vesicle) exocytosis in neuroendocrine PC12 cells. This indicates that each of these proteins is distinct in its mechanism for assembling SNARE complexes. We will determine the basis for these differences using a series of novel assays for SNARE complex assembly in Aim 1. The assays take advantage of our success in generating full-length active proteins for biochemical studies. Completion of this work will provide a new view of SNARE regulatory proteins, significantly impacting our understanding of the molecular basis for neuropeptide and peptide hormone secretion. We have found that two other proteins in the Munc13 family, Munc13-4 and BAIAP3, act as tethering/priming factors for intracellular membrane fusion events. Ubiquitously-expressed Munc13-4 was required for the merge of a subset of endosomal compartments. In neuroendocrine cells, Munc13-4+ endosomes also interacted with DCVs in a novel process that will be elucidated in Aim 2. Neural/endocrine- specific BAIAP3 is required for evoked peptide secretion in neuroendocrine cells but localizes to the late Golgi and to a small subset of DCVs.
In Aim 3, we will determine if BAIAP3 functions in the homotypic fusion events that characterize DCV maturation. For both Munc13-4 and BAIAP3, we will determine their sites of action, define the fusion events they promote, and identify the SNARE proteins they regulate. This work will characterize intracellular membrane fusion events that currently have no known regulators, and will reveal novel points of intersection between the endosomal and regulated secretory pathways in neuroendocrine cells. It will also impact the understanding of pathologies linked to mutations in Munc13-4 and BAIAP3.

Public Health Relevance

Numerous genetic diseases result from mutations in proteins that control membrane trafficking in cells, and these frequently affect neural function. Different members of protein families can control distinct steps on the inbound (endosomal) or outbound (secretory) branches of trafficking pathways. The goal of this research is to understand the roles of members of a Munc13 protein family that operate in both branches so that there is a fuller understanding of genetic diseases that are linked to this protein family.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK025861-38
Application #
9229547
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Malozowski, Saul N
Project Start
1979-07-01
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
38
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Zhang, Xingmin; Jiang, Shan; Mitok, Kelly A et al. (2017) BAIAP3, a C2 domain-containing Munc13 protein, controls the fate of dense-core vesicles in neuroendocrine cells. J Cell Biol 216:2151-2166
Woo, Sang Su; James, Declan J; Martin, Thomas F J (2017) Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles. Mol Biol Cell 28:792-808
Chehab, Tarek; Santos, Nina Criado; Holthenrich, Anna et al. (2017) A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel-Palade body exocytosis in endothelial cells. Mol Biol Cell 28:1688-1700
Kabachinski, Greg; Kielar-Grevstad, D Michelle; Zhang, Xingmin et al. (2016) Resident CAPS on dense-core vesicles docks and primes vesicles for fusion. Mol Biol Cell 27:654-68
Petrie, Matt; Esquibel, Joseph; Kabachinski, Greg et al. (2016) The Vesicle Priming Factor CAPS Functions as a Homodimer via C2 Domain Interactions to Promote Regulated Vesicle Exocytosis. J Biol Chem 291:21257-21270
Martin, Thomas F J (2015) PI(4,5)P?-binding effector proteins for vesicle exocytosis. Biochim Biophys Acta 1851:785-93
Yamaga, Masaki; Kielar-Grevstad, D Michelle; Martin, Thomas F J (2015) Phospholipase C?2 Activation Redirects Vesicle Trafficking by Regulating F-actin. J Biol Chem 290:29010-21
Kabachinski, Greg; Yamaga, Masaki; Kielar-Grevstad, D Michelle et al. (2014) CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis. Mol Biol Cell 25:508-21
James, Declan J; Martin, Thomas F J (2013) CAPS and Munc13: CATCHRs that SNARE Vesicles. Front Endocrinol (Lausanne) 4:187
Zhang, Zhao; Takeuchi, Hiroshi; Gao, Jing et al. (2013) PRIP (phospholipase C-related but catalytically inactive protein) inhibits exocytosis by direct interactions with syntaxin 1 and SNAP-25 through its C2 domain. J Biol Chem 288:7769-80

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