Regulated exocytosis is a stimulus-dependent membrane fusion event of fundamental importance to a range of physiological processes. The membrane fusion reaction involves substantial lipid rearrangements and is opposed by the powerful hydrophobic force. For fusion to occur, a high energy barrier must be overcome. The overall goal of this proposed research is to determine the functional roles of membrane bilayer remodeling in overcoming the energy barrier of exocytic vesicle fusion, using the trafficking of the glucose transporter GLUT4 as a model system. To achieve this goal, we will employ a unique combination of complementary approaches including biochemical reconstitution, electron microscopic imaging, and biophysical measurements. First, we will define how two membrane bilayers are brought into close apposition by the SNARE-SM complex to prepare for the subsequent steps of membrane remodeling and merging. Next, we will assess the functional role of vesicle membrane bending in the fusion reaction. Finally, we will examine how the fusion reaction is regulated by local remodeling of the plasma membrane. We hypothesize that the fusion of exocytic vesicles with the plasma membrane involves a novel dual-curvature-induction mechanism: while the amphipathic motif of the v-SNARE bends the vesicle membrane, the hydrophobic loops of C2-domain molecules penetrate into the plasma membrane and induce local membrane curvature. Together, these membrane-bending activities are expected to create curvature stresses at the fusion sites to overcome the energy barrier for the fusion reaction. Successful completion of this proposed research will provide key insights into the molecular mechanisms of exocytic vesicle fusion. This work will also serve as a paradigm for understanding the general principles of intracellular membrane fusion. Ultimately, our findings will facilitate the development of novel therapeutic strategies for diseases associated with dysfunctional regulated exocytosis including diabetes, epilepsy, and immune disorders.

Public Health Relevance

Regulated exocytosis is critical to the cell surface localization of receptors and transporters that are key to human physiology. We will investigate membrane dynamics and remodeling in the exocytic vesicle fusion process. Imbalances in receptor/transporter exocytosis give rise to severe forms of human disease. This study will provide key insights into these diseases and may offer new strategies for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM102217-02
Application #
8641404
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2013-04-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
2
Fiscal Year
2014
Total Cost
$260,775
Indirect Cost
$89,775
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309