FYVE2, a phosphatidylinositol 3-phosphate effector, interacts with the COPII machinery to control autophagosome formation in Arabidopsis

Abstract

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase SAR1, which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.

Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole

“…Our findings are consistent with a role in autophagosome maturation as opposed to autophagosome biogenesis as was recently suggested (Kim et al, 2022): (i) our differential centrifugation-based autophagosome enrichment procedure selects for proteins that associate with closed, mature autophagosomes. AP-MS did not identify biogenesis components but did identify trafficking related proteins such as Myosin 14 (Fig 1B, Table S3), (ii) we observe fully formed autophagosomes in micrographs obtained from cfs1 mutants indistinguishable from and in similar numbers to wild type (Fig.…”

“…1B-C). Since CFS1 has previously been linked to autophagy and FYVE and SYLF domain-containing proteins are well-known players in vesicle trafficking (Sutipatanasomboon et al, 2017; Kim et al, 2022; Melia et al, 2020), we decided to characterize CFS1 in depth.…”

“…We developed a differential centrifugation protocol to enrich for intact autophagosomes prior to affinity purification-mass spectrometry (AP-MS) with ATG8 as bait. This approach identified CFS1 ( c ell death related endosomal F YVE/ S YLF protein 1), a highly conserved FYVE (Fab-1, YGL023, Vps27, and EEA1) and SYLF (SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE) domain-containing protein that was previously linked to autophagy (Sutipatanasomboon et al, 2017; Kim et al, 2022). Characterization of CFS1 revealed that it interacts with ATG8 in an AIM-dependent manner and specifically regulates autophagic flux in both Arabidopsis thaliana and Marchantia polymorpha .…”

Monitoring Autophagy in Rice With GFP-ATG8 Marker Lines

“…Repeated identification of the same proteins actually added more confidence to the IP-MS study. A number of proteins identified are worthy of further study and may help answer some existing questions in plant autophagy, such as which membranes or vesicles, apart from COPII vesicles ( Zeng et al, 2021 ; Kim et al, 2022 ), may contribute to autophagosome formation. Early studies for plant autophagy had identified ATI1 and ATI2 as plant-specific ATG8-interacting proteins; both were uncovered through an Y2H screen ( Honig et al, 2012 ).…”

COPII vesicles in plant autophagy and endomembrane trafficking

“…Plant cells contain not only canonical Atg components, but also plant‐specific proteins functioning in autophagosome formation [21–23]. Recent studies have demonstrated the contribution of COPII vesicles to the autophagy pathway in plants [24,25]. In this review, we summarize the roles of plant COPII vesicles in canonical and non‐canonical ER export and recap known facts of plant autophagy interplaying with the endomembrane system.…”