Gookin, T. E. | Search bioRxiv

bioRxiv2023

Influence of expression protocols on Gα biochemical activity: kinetics of plant and mammalian G protein cycles

Heterotrimeric G proteins are a class of signal transduction complexes with broad roles in human health and agriculturally important plant traits. In the classic paradigm, guanine nucleotide binding to the G subunit regulates the activation status of the complex. Using the Arabidopsis thaliana G subunit, GPA1, we developed a rapid StrepII-tag mediated purification method that facilitates isolation of protein with increased enzymatic activities as compared to conventional methods, and is demonstrably also applicable to mammalian G subunits. We subsequently utilized domain swaps of GPA1 and human GNAO1 to demonstrate the instability of recombinant GPA1 is a function of the interaction between the Ras and helical domains, and can be partially uncoupled from the rapid nucleotide binding kinetics displayed by GPA1.

biochemistry↗

bioRxiv2020

A G protein-coupled receptor-like module regulates Cellulose Synthase secretion from the endomembrane system in Arabidopsis

Cellulose synthesis is essential for plant morphology, water transport and defense, and provides raw material for biomaterials and fuels. Cellulose is produced at the plasma membrane by Cellulose Synthase (CESA) protein complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked from the Golgi apparatus and trans-Golgi Network (TGN) to the plasma membrane. Since CESA enzymes are only active in the plasma membrane, control of CSC secretion is a critical step in the regulation of cellulose synthesis. However, the regulatory framework for CSC secretion is not clarified. In this study, we identify members of a family of seven transmembrane domain-containing proteins (7TMs) as important for cellulose production during cell wall integrity stress. 7TM proteins are often associated with guanine nucleotide-binding protein (G) protein signalling and mutants in several of the canonical G protein complex components phenocopied the 7tm mutant plants. Unexpectedly, the 7TM proteins localized to the Golgi apparatus/TGN where they interacted with the G protein complex. Here, the 7TMs and G proteins regulated CESA trafficking, but did not affect general protein secretion. Furthermore, during cell wall stress, 7TMs localization was biased towards small CESA-containing vesicles, specifically associated with CSC trafficking. Our results thus outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses upon exposure to cell wall integrity stress.

plant biology↗