Domain interactions mediating membrane association of GBF1-type plant ARF-GEFs – mechanistic and evolutionary analysis

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The subcellular distribution of proteins by membrane traffic plays an important role in developmental and physiological processes of eukaryotes. The formation of transport vesicles requires activation of small ARF GTPases through GDP-GTP exchange which is catalysed by ARF guanine-nucleotide exchange factors (ARF-GEFs). Large ARF-GEFs like GBF1 in mammals or GNOM in plants comprise 6 conserved domains: The N-terminal DCB domain (dimerisation) is joined by HUS, SEC7 (GDP-GTP exchange) and HDS1 to HDS3; of these domains, HUS and HDS1-3 domains have not been functionally characterised. In mammalian GBF1, the DCB domain interacts with the adjacent HUS domain. By contrast, in GBF1-related GNOM, the DCB domain interacts with the complementary deltaDCB fragment – the three domains HUS, SEC7 and HDS1 are required – to enable membrane association of GNOM. The difference in domain interaction correlates with a difference in the subcellular site of action between the two ARF-GEFs. GBF1 acts at the Golgi apparatus, mediating the formation of COPI vesicles for the retrograde transport to the ER, whereas GNOM has a role at endosomes, being required for the polar recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane. The aim of this proposal is to identify the mechanism(s) of domain interaction mediating the membrane association of GNOM. We will pursue two approaches which both involve yeast interaction experiments and functional studies in Arabidopsis. In an evolutionary strategy, ARF-GEFs from different eukaryotic species will be analysed to determine where the boundary between general GBF1-type ARF-GEFs and plant-specific GNOM-type ARF-GEFs might run across the eukaryotes. The anticipated results also have practical implications. In order to gain insights into mechanisms of domain interaction of GNOM, we will analyse chimeric proteins to which GNOM and a most closely related general GBF1-type ARF-GEF contribute complementary segments. This will be done to minimise the risk of potential incompatibilities resulting from vast evolutionary distances. The aim of this second approach is to identify the relevant sequence motifs that enable the DCB-deltaDCB interaction of GNOM. The experiments planned might be supported by a structural model of GNOM that is being developed. In addition to these directed alterations, we propose a functional assay for the identification of interaction faces that is based on random mutagenesis of GNOM domains. Here, a population of mutagenised domains will be selected for the loss of DCB-deltaDCB interaction; the mutant domains will be checked for full length to make sure that they have merely lost their ability for interaction between the complementary parts.