Autophagosome biogenesis in plants

Abstract

The autophagy core machinery is essentially conserved in eukaryotic cells for autophagy regulation. However, the underlying mechanisms for autophagosome formation in plant cells remain elusive. We have recently demonstrated that SH3 domain-containing protein 2 (SH3P2), a BAR (Bin-Amphiphysin-Rvs) domain protein, functions as a novel regulator for autophagosome biogenesis in Arabidopsis thaliana. Using SH3P2 and its GFP fusion as probes, we have characterized the dynamics and structures of autophagosome formation in plant cells. The phagophore assembly site, marked by SH3P2, is identified as having a close connection with the ER. SH3P2 also binds to phosphatidylinositol 3-phosphate (PtdIns3P) and functions downstream of the phosphatidylinositol 3-kinase (PtdIns3K) complex. Thus, SH3P2 serves as a novel membrane-associated protein in regulating autophagosome formation in Arabidopsis thaliana.

The autophagosome is a unique double-membrane organelle that is formed during autophagy and functions in delivering cargo to the vacuole for degradation. Autophagosome formation involves multiple curved structures for its drastic morphological changes: the phagophore assembly site (PAS); omegasomes; completed autophagosomes with a bi/multi-layer membrane; amphisomes; and autophagic bodies in the vacuole. In yeast and animal cells, macroautophagy is mainly regulated by the autophagy core machinery, which shares the conserved autophagy-related (ATG) proteins.

In our recent study, we determined that SH3-domain containing protein 2 (SH3P2/AT4G34660), a BAR domain protein, functions as a novel regulator for autophagosome formation in Arabidopsis thaliana. Upon autophagy induction, SH3P2 translocates to numerous punctate structures that are colocalized with autophagosome markers including ATG8 and ATG6.

To further understand in which step(s) SH3P2 is involved during autophagy, we have characterized the dynamics of SH3P2-GFP in transgenic Arabidopsis plants. Upon autophagy induction, crescent- and ring-like structures labeled by SH3P2-GFP are clearly detected, and tubular structures marked with this chimera are also seen. These observations are similar to the well-defined process of autophagosome formation in yeast and animal cells. We further examined the nature of these SH3P2-positive structures via immunogold electron microscopy using SH3P2 antibodies. SH3P2 is readily detected along the membrane of bi/multi-layer structures. Thus, these data indicate that SH3P2 is distributed on the autophagosome membrane, and the process for autophagosome membrane progression is conserved in Arabidopsis thaliana (Fig. 1). Interestingly, we found that a portion of the PAS labeled by SH3P2 exhibits evident ribosomes or ER fragments on both the outside and inside membrane surfaces, suggesting that these SH3P2-positive structures are likely derived from the ER membrane. Conversely, we also observed that some SH3P2-GFP-positive autophagosome structures do not exhibit a direct connection to the ER. Future efforts are needed to verify whether other membrane sources, such as mitochondria and the Atg9 reservoir that have been implicated in either mammalian or yeast cells, contribute to the generation of the PAS in plants (Fig. 1).

Figure 1. Possible roles of SH3P2 and its interacting complex in autophagy and autophagosome formation in Arabidopsis. In this working model, SH3P2 traffics to the phagophore/PAS and is probably responsible for membrane deformation from various possible membrane sources (e.g., ER, mitochondria, etc. as indicated by question marks). SH3P2 associates with the PtdIns3K complex via a yet-to-be identified factor (box with question mark) and actively participates in the expansion and maturation steps during autophagosome formation in plants. For comparison, the current model of the PtdIns3K complex and its known components in regulating autophagosome formation in mammals are also shown in the right box.

Further analysis using SH3P2 RNAi transgenic Arabidopsis plants shows that autophagy requires the activity of SH3P2, because knockdown of SH3P2 suppresses autophagosome formation, and SH3P2 may function during the expansion or maturation step during autophagosome biogenesis. Unlike most of the ATG genes that have been reported in plants so far, downregulation of SH3P2 results in a lethality defect in Arabidopsis. Interestingly, in our yeast-2 hybrid and immunoprecipitation assays, we showed that SH3P2 may interact with ATG8 through the SH3 domain. Although the biological significance of this interaction remains elusive, it is possible that SH3P2 is targeted by ATG8 for degradation or recycling in the vacuole, which in turn may regulate the progression of autophagy.

Membrane-remodeling proteins ATG14 and SH3GLB1/Bif-1 have been identified for the regulation of autophagosome formation in yeast or mammalian cells. However, homologs of these key regulators have not been identified in plants. The BAR domain is a well-defined membrane module for membrane deformation. Interestingly, SH3P2 contains a BAR domain at its N terminus, which is structurally similar to that in SH3GLB1. Our membrane/lipid binding data confirm that SH3P2 is membrane-associated and preferentially binds to PtdIns3P. In addition, SH3P2 is associated with the PtdIns3K complex, and treatment with the PtdIns3K inhibitor wortmannin blocks the translocation of SH3P2 to the phagophore or autophagosome membrane, implying that SH3P2 is downstream of the PtdIns3K complex. In mammalian cells, SH3GLB1 interacts with the PtdIns3K complex via UVRAG to regulate autophagy. It is reported that local PtdIns3P generation is a prerequisite for PAS formation, which is achieved by the PtdIns3K complex. Based on our data, it is very likely that SH3P2 also interacts as part of the PtdIns3K complex to mediate autophagosome formation in Arabidopsis thaliana. Figure 1 shows a working model for the possible roles of SH3P2 in mediating autophagosome biogenesis in plants; however, it is necessary to identify the missing link between SH3P2 and the PtdIns3K complex in future investigations.

Zhuang X, Wang H, Lam SK, Gao C, Wang X, Cai Y, Jiang L. A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis. Plant Cell. 2013;25:4596–615. doi: 10.1105/tpc.113.118307.