AtNBR1 Is a Selective Autophagic Receptor for AtExo70E2 in Arabidopsis.

Selective autophagy is a subcellular process whereby cytoplasmic materials are selectively sequestered into autophagosomes for subsequent delivery to the vacuole for degradation and recycling. Arabidopsis (Arabidopsis thaliana) NBR1 (next to BRCA1 gene 1 protein; AtNBR1) has been proposed to function as a selective autophagy receptor in plants, whereby AtNBR1 anchors the ubiquitinated targets to autophagosomes for degradation. However, the specific cargos of AtNBR1 remain elusive. We previously showed that Arabidopsis exocyst subunit EXO70 family protein E2 (AtExo70E2), a marker for exocyst-positive organelle (EXPO), colocalized with the autophagosome marker Arabidopsis autophagy-related protein8 (AtATG8) and was delivered to the vacuole for degradation upon autophagic induction. Here, through multiple analyses, we demonstrate that AtNBR1 is a selective receptor for AtExo70E2 during autophagy in Arabidopsis. First, two novel loss-of-function nbr1 CRISPR mutants (nbr1-c1 and nbr1-c2) showed an early-senescence phenotype under short-day growth conditions. Second, during autophagic induction, the vacuolar delivery of AtExo70E2 or EXPO was significantly reduced in nbr1 mutants compared to wild-type plants. Third, biochemical and recruitment assays demonstrated that AtNBR1 specifically interacted and recruited AtExo70E2 or its EXPO to AtATG8-positive autophagosomes in a ubiquitin-associated (UBA)-independent manner during autophagy. Taken together, our data indicate that AtNBR1 functions as a selective receptor in mediating vacuolar delivery of AtExo70E2 or EXPO in a UBA-independent manner in plant autophagy.

Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants.

Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner.