Autophagy and Vesicle Trafficking in Arabidopsis: Emerging Roles of Gamma Secretase Complex Subunits and AP4 Complex

Abstract

Presenilins are the catalytic component of γ-secretase complex which was first identified in the genetic screens of the patients suffering from the Alzheimer's disease. Macroautophagy is a major route which encompasses degradation of the cell components and damaged proteins, lipids and carbohydrates to recycle nutrients in response to starvation. Here we report that Arabidopsis presenilin double mutant <em>ps1/ps2</em> shows defective clearance of autophagosomes in root cells when autophagy is induced through sucrose starvation. In addition to being susceptible to the sucrose starvation, mutant root cells have altered expression levels of important autophagy-related genes. Mutant root cells show differential protein accumulations under sucrose starvation leading to inefficient clearance of proteins. Biochemical and cell biological approaches combined with autophagy inhibitors such as wortmannin and concanamycin-A suggested aberrant degradation of autophagosomes within the lytic plant vacuoles. Taken together, our data suggest an involvement of the γ-secretase complex and/or presenilins in plant autophagy. Presenilin enhancer-2 (PEN2) is another subunit of the γ-secretase complex which was first discovered in a genetic study involving <em>C. elegans</em>. It is required for the γ-secretase complex activity and undertakes the endoproteolysis of presenilins. Here, we demonstrate that AtPEN2 vesicles are very sensitive to latrunculin-B, an F-actin depolymerizing drug, which suggests the role of the actin cytoskeleton in the motility of these vesicles. Moreover, AtPEN2 partially localizes with DsRED-FYVE, a PI3P reporter, which is specifically localized with the dynamic and highly motile late endosomal compartments and has been implicated in the tip growth. Furthermore, the phenotypic analysis of pen2 mutant reveals reduced primary root growth compared to the wild type seedlings. Collectively, our results indicate possible roles of AtPEN2 in regulating tip growth and protein trafficking pathways in Arabidopsis. However, studies on other possible functions of AtPEN2 in signal transduction and stress responses are still required. Adaptor protein (AP) complexes are conserved throughout eukaryotic organisms and are vital for protein sorting among various post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP1-AP5), AP4 is the most poorly understood. In animals, AP4 has recently been recognized as a regulator of autophagy through mediating export of ATG9, a core autophagy protein from the trans-Golgi to promote autophagosome formation. Here we have performed an analysis of Arabidopsis mutants lacking different subunits of AP4 in connection with autophagy. We report that the YXXØ motif is conserved in the ATG9 protein of Arabidopsis which is required for its recognition by AP4 complex. Moreover, colocalization study reveals that AP4 complex localizes with ATG9 in the <em>Nicotiana benthamiana</em> leaf epidermal cells. Besides showing sensitivity towards dithiothreitol (DTT), an ER stress inducer, the mutants of AP4 complex accumulate ATG8, a structural component of autophagosomes. Taken together, we propose that Arabidopsis AP4 complex may interact with ATG9 and play a role in its transport to the phagophore assembly site similar to animals and in addition to missorting of proteins, defective autophagy is also responsible for the phenotypic abnormality of AP4 mutants.