Comparative analysis of a membrane trafficking regulator in flowering plants

Abstract

In eukaryotic cells, membrane traffic is essential for the preservation of protein transport, cell-cell communication, pathogen defense and other pivotal processes in plant life. The delivery of cargo molecules occurs in vesicles that bud from a donor compartment, followed by transport along the cytoskeleton, membrane tethering and fusion with the target membrane. Vesicle formation is initiated through the activation of ADP-ribosylation factors (ARFs) by their guanine-nucleotide exchange factors (ARF-GEF). The two closely-related ARF-GEFs GNOM and GNL1 in Arabidopsis thaliana both mediate retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER). Interestingly, GNOM has an additional task in endosomal recycling of the auxin efflux carrier PIN1 to the basal plasma membrane. This functional diversification of GNOM and GNL1 resulted in different subcellular localizations of the two proteins. While GNOM is recruited to the putative recycling endosome, GNL1 localizes to the Golgi. We aimed to identify protein domains conferring membrane specificity by expressing GNOM/GNL1 chimera in the gnom mutant background. These rescue experiments led us hypothesize that critical motifs for the endosomal recruitment are exposed on the protein surface of GNOM through the interaction between three adjacent domains (HUS, SEC7 and HDS1). In addition, we investigated the origin of the functional diversification. By expressing GNOM and GNL1 homologs from different Viridiplantae species in the gnom and gnl1 mutant background, we reason that the endosomal PIN1 recycling is an ancient function that was passed on over time and that eventually got lost during the evolution of GNL1. Previous yeast two-hybrid studies in our lab revealed that the functional diversification resulted in the prevention of GNOM-GNL1 heterodimers. We used the same assay to test GNOM/GNL1-related proteins from different species for their ability to heterodimerize. Our preliminary results suggest that the functional diversification was initiated within the eurosids of the dicotyledonous flowering plants. GNOM dimerization is dependent on the homotypic interaction between two DCB domains. Yeast two-hybrid studies revealed that the positively charged amino acid lysine at position 120 mediates DCB-DCB interaction. Future experiments in Arabidopsis expressing the DCB domain with the K120D mutation could identify the significance of GNOM dimerization.