Investigation of IRQ domain containing proteins in Arabidopsis thaliana

Abstract

The endomembrane system in eukaryotic cells plays a vital role in the movement of membranes and substances around the cell in response to abiotic and biotic stimuli. Recent work on an actin binding protein, NETWORKED4B (NET4B), revealed that this vacuolar localised protein (Deeks et al. 2012) contains a domain termed the IRQ domain responsible for interacting with particular regulatory proteins of the endomembrane system. Bioinformatic analysis revealed that this IRQ domain was present in six novel proteins not containing the characteristic NET Actin Binding (NAB) domain. These proteins were termed the IRQ proteins (IRQ1-6). Work outlined in this thesis explores the evolution and localisation of expression of these proteins but in particular looks at IRQ4. Phylogenetic analysis revealed that the IRQ proteins represent a eudicot specific group of proteins and that they evolved from the NET proteins. The IRQ proteins can be subdivided based on sequence similarity into three groups: IRQ1 and IRQ6, IRQ2 and IRQ3, IRQ4 and IRQ5. Using promoter GUS lines for IRQ1 and IRQ6 revealed that these proteins may be involved in the initiation or regulation of lateral root growth. IRQ4 was expressed most strongly in the root. Subcellular localisation analysis using promIRQ4::IRQ4-GFP and live cell imaging showed that IRQ4 localises to the prevacuolar compartment (PVC)/multivesicular body (MVB). Immunogold labelling using an IRQ4 specific antibody revealed an additional localisation to autophagasomes. This project investigates a group of novel eudicot specific proteins and shows that IRQ4 may be involved in key endomembrane pathways in plants.