HAPLESS13, The Arabidopsis μ1 Adaptin, Is Essential For Protein Sorting At The Trans-Golgi Network/Early Endosome

Plant cells contain sophisticated endomembrane compartments, intracellular protein sorting between the various locations in the endomembrane system occurs in both secretory and endocytic routes. Vesicles in the secretory route start at the endoplasmic reticulum, passing through the Golgi before reaching the trans-Golgi network(TGN)/early endosome(EE). The TGN/EE in Arabidopsis(Arabidopsis thaliana) is an independent and highly dynamic organelle. Once reaching the TGN/EE, proteins delivered by their vesicle carriers are subject to further sorting, being incorporated either into vesicles that pass through the PVC/MVB before reaching the vacuole for degradation or into vesicles that enter the secretory pathway for delivery to the PM.Therefore, the TGN/EE is a critical sorting compartment that lies at the intersection of the secretory and endocytic routes.Intracellular protein sorting relies on sorting signals within cargo proteins and on the molecular machinery that recognizes sorting signals. Adaptor proteins(AP) play a key role in the recognition of sorting signals. APs are heterotetrameric protein complexes composed of two large subunits(γ and β), a small subunit(σ), and a medium subunit(μ) that is crucial for cargo selection. APs associate with the cytoplasmic side of secretory and endocytic vesicles, recruiting coat proteins and recognizing sorting signals within cargo proteins for their incorporation into vesicle carriers. AP1M2 was previously named HAPLESS13(HAP13), whose mutant allele hap13 showed male gametophytic lethality. In recent quests for AP-1 in plants, HAP13/AP1M2 was con?rmed as the Arabidopsis μ1 adaptin based on its interaction with other components of the AP-1 complex as well as its localization at the TGN. A novel mutant allele of HAP13/AP1M2, ap1m2-1, was found to be defective in the intracellular distribution of KNOLLE, leading to defective cytokinesis. However, it was not clear whether HAP13/AP1M2 mediated other cellular activities and their developmental consequences.The main conclusions presented in this thesis are as follows:(1) Cell morphogenesis was disturbed in hap13-1.To determine the function of HAP13 in sporophytic tissues, we characterized an additional allele of HAP13 and con?rmed that no full-length transcript of HAP13 could be detected in hap13-1/ap1m2-1(Teh et al., 2013). However, a partial transcript potentially encoding a truncated HAP13 with the ?rst 229 amino acids(HAP13N229) was detected in hap13-1. hap13-1 showed pleiotropic developmental defects, growth dwarfed, the growth of primary roots arrested precociously, hardly made the transition to reproductive growth when grown in soil. Leaf pavement cells were signi?cantly smaller and lost their characteristic jigsaw puzzle shape in hap13-1.Trichomes are less complex in hap13-1, most trichomes on hap13-1 leaves showed only one branch. Defects in cell morphogenesis in hap13-1 extended to the epidermal cells in roots and hypocotyls, the growth polarity of hap13-1 cells was disrupted, leading to irregularly shaped or ballooning cells, root hairs were either bulged or hardly initiated in hap13-1.(2) hap13-1 was insensitive to BFA washout, indicative of defects in guanine nucleotide exchange factors for ADP-ribosylation facto(ArfGEF) mediated post-Golgi traf?cking.To determine which traf?cking routes are regulated by HAP13,we used the endocytic tracer FM4-64 and BFA in an uptake study. FM4-64 entry from the PM was signi?cantly increased in hap13-1 at various time points, as represented by the number of endosomes. Treatment with 50 mM BFA resulted in intracellular aggregation of FM4-64 signals in the wild type, while BFA washout redistributed FM4-64 from the intracellular aggregates(BFA compartments) to endosomes. By contrast, a signi?cantly larger amount of the FM4-64 signal remained at endosomes upon BFA treatment in hap13-1. A large amount of the FM4-64 signal was still enclosed in BFA compartments beside endosome signals upon BFA washout.(3) Functional loss of HAP13(hap13-1/ap1m2-1) resulted in a full spectrum of growth defects, suggestive of compromised auxin signaling and of defective RLK signaling.To further dissect vesicle traf?cking routes in which HAP13 plays critical roles, we introduced ?uorescent protein fusions of potential cargos in hap13-1 and analyzed their dynamic subcellular distribution. PIN2:GFP was distributed asymmetrically in the epidermal cells of wild-type roots. By contrast, the distribution of PIN2:GFP in hap13-1 was irregular at the PM. A higher percentage of BRI1 signals were detected as punctate vesicles in hap13-1 than in the wild type. BFA caused an aggregation of BRI1:YFP and PIN2:GFP to the BFA compartments in hap13-1. A higher percentage of BRI1 and PIN2 signals were still present in the BFA compartments and in the cytoplasm in hap13-1 after BFA washout.(4) HAP13/AP1M2 showed evolutionarily conserved function during vacuolar fusion, providing additional support to its identity as a μ1 adaptin.To further con?rm that HAP13 is the bona ?de μ-subunit of AP-1, we introduced HAP13 into the yeast amp1- mutant. HAP13 complemented the vacuolar fusion defect of the yeast amp1- mutant upon osmotic stress.In summary, these results demonstrate the importance of the Arabidopsis μ1 adaptin for intracellular protein sorting centered on the TGN/EE.