Imoukhuede, Princess Izevbua (2008) Visualizing the membrane confinement, trafficking and structure of the GABA transporter, GAT1. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-03142008-155402
Transporter trafficking regulators can play an important role in maintaining the transporter density necessary for effective function. I determine interactions that confine GAT1 at the membrane by investigating GAT1 lateral mobility through fluorescence recovery after photobleaching (FRAP). I find that the mobility of GAT1 can be increased by depolymerizing actin or by blocking the GAT1 PDZ interacting domain. I also identify ezrin as the GAT1 adaptor to actin. Through fluorescence resonance energy transfer (FRET), the distance between GAT1-YFP and Ezrin-CFP is calculated as 64--68 Å, and it can be significantly increased by disrupting the actin cytoskeleton. Altogether, my data reveals that actin confines GAT1 to the plasma membrane via ezrin, an interaction mediated through the GAT1-PDZ interaction domain. Discoveries in the field of vesicle fusion provide direct ties to translational research. While the study of vesicle fusion classically has been applied to neurotransmitter and neuropeptide containing vesicles; there is evidence that secretory vesicles physiologically differ from vesicles trafficking membrane protein. For instance, GAT1 resides on a vesicle lacking neurotransmitter but containing some v-SNARE proteins. These differences in the vesicle composition suggest inherent differences in trafficking mechanisms, which can only be confirmed through further study of membrane protein trafficking. To this end, I apply total internal reflection fluorescence microscopy (TIRFM) to quantify the number of GAT1 molecules on vesicles and to observe the movement of vesicles containing fluorescently tagged GAT1 into the plasma membrane. I determine that these vesicles contain 3--7 molecules of GAT1 and uncover a population of GAT1 vesicles with ATP-dependent lateral displacement. The protein-protein interactions, trafficking, and oligomerization of mouse GAT1 were studied using fourteen different fusions of mGAT1 with fluorescent protein. We determine that a natural PDZ-interacting motif is minimally required for wild-type GAT1 behavior. Fusions with wild-type function yielded up to 21% FRET efficiency, indicating efficient GAT1 oligomerization. Additionally, 45% FRET was observed between a GAT1 construct and YFP-syntaxin-1A. Inserting XFP between R565 and L566, resulted in 33% FRET but impaired function, which indicated the “RL” motif in the proximal C terminus governs export from the endoplasmic reticulum but not transporter oligomerization.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||fluorescence; FRAP; FRET; GABA transporter; GAT1; microscopy; TIRF; trafficking|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||8 February 2008|
|Non-Caltech Author Email:||princess (AT) alum.mit.edu|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||31 Jul 2008|
|Last Modified:||10 Dec 2014 20:20|
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