Heterotrimeric G-proteins: new analytical tools and identification of novel signaling components
Open Access
- Author:
- Gookin, Tim Elliott
- Graduate Program:
- Plant Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 05, 2015
- Committee Members:
- Sarah Mary Assmann, Dissertation Advisor/Co-Advisor
Sarah Mary Assmann, Committee Chair/Co-Chair
John Edward Carlson, Committee Member
Gabriele Brigitte Monshausen, Committee Member
Andrew George Stephenson, Committee Member - Keywords:
- G proteins
GPCR
prediction
receptor
signaling
protein-protein interaction
BiFC
leaf death
Arabidopsis - Abstract:
- Heterotrimeric G-protein signaling is one of the most important mechanisms eukaryotic organisms use to sense their environment, and as such, it is one of the most studied molecular signaling systems in eukaryotic biology. In humans, the intracellular heterotrimeric G-protein complexes play critical roles in our sense of sight, taste, smell, emotion,and central physiology by receiving extracellular signals perceived and transduced by plasma-membrane localized seven transmembrane G-protein coupled receptors (GPCRs). These numerous and diverse physiological processes are facilitated by > 800 predicted and known GPCRs in humans which signal to a large number of G-protein compexes with a theoretical combinatorial complexity of over 900 configurations. Similar to metazoan organisms, plants also sense and respond to their environment and it is abundantly clear that plants propagate a numerous and diverse set of signals through the hetrotrimeric G-protein complex. But in plants the number of known G-protein complex subunits is greatly reduced. There are no confirmed GPCRs, and prior to finishing this dissertation, the number of Arabidopsis G-protein complex configurations was limited to three. The question remains as to how the relatively sparse repertoire of plant G-proteins can be responsible for the wide range of physiological processes affected by G-protein subunit mutation. In this work: 1) I detail the bioinformatic identification and empirical validation of new candidate GPCRs, 2) develop and improve the Bimolecular Fluorescence Complementation methodology for in-planta protein-protein interaction, 3) use this new methodology in conjunction with other experimental methods to show the actual number of plant G-protein subunits is greater than previously thought, and 4) characterize two homologous candidate GPCRs. While it appears certain the two proteins analyzed are not plant Gα-coupling GPCRs, they do play critical role in maintaining metabolic homeostasis; double mutants exhibit developmental stage-specific catastrophic leaf death in response to short photoperiod.