A novel reticulophagy receptor, Epr1: a bridge between the phagophore protein Atg8 and ER transmembrane VAP proteins

ABSTRACT

Reticulophagy, a type of selective autophagy that specifically targets and degrades parts of the endoplasmic reticulum (ER) network (sheets or tubules), plays a crucial role in the responses to ER stress. The selectivity of the ER cargo recognition relies on the unique reticulophagy receptors, which tether and deliver cargos to phagophores, the precursors to autophagosomes. Various integral membrane proteins have been well characterized as reticulophagy receptors, including Atg39, Atg40, RETREG1/FAM134B, SEC62, RTN3L, CCPG1, TEX264, and ATL3, in both yeast and mammals in the past five years. In a recent paper, Zhao et al. discovered in fission yeast a novel reticulophagy receptor, Epr1, which bridges the ER and phagophore by binding to Atg8 and VAPs, a mechanism different from the aforementioned reticulophagy receptors.

KEYWORDS:

• Autophagy
• endoplasmic reticulum
• stress
• vacuole
• yeast

Macroautophagy can be divided into selective and nonselective types of autophagy depending on whether or not it involves high specificity in the choice and delivery of cargo for degradation [1]. There are three ways for selective autophagy receptors to coordinate cargos with phagophores [2]. In the type 1 connection, receptors (e.g., Atg19 in the yeast cytoplasm-to-vacuole targeting pathway) interact directly with a specific protein component of a cargo; the type 2 receptor (e.g., SQSTM1 in mammalian cells) recognizes a ubiquitin modification on a cargo; and with type 3, the receptor (e.g., Atg32 in yeast mitophagy) is an integral membrane protein embedded in the outer membrane of a cargo organelle. Previously, the known reticulophagy-specific autophagy receptors were all integral ER membrane proteins (type 3) [3]. In contrast to these earlier studies, the novel reticulophagy receptor Epr1 characterized by Zhao et al. allows phagophores and the ER to interact through a type 1 connection [4]. Prior to this paper, the only known type 1 soluble autophagy receptors were Komagataella phaffii/Pichia pastoris Atg30 and S. cerevisiae Atg36, which are pexophagy receptors [5].

In the study, the authors also revealed a novel role of vesicle-associated membrane protein-associated proteins (VAPs) in reticulophagy regulation. VAPs have multiple roles in maintaining ER morphology, function and cell signaling [6]. The primary role of VAPs is to serve as a recruiting platform for anchoring soluble cytosolic proteins to the outer surface of the ER. In mammalian cells, VAPs interact with subunits of the ULK1 complex to promote autophagosome biogenesis [7]. VAPs are also important for maintaining the ER-plasma membrane (PM) contact in yeast. The investigation of the roles of Epr1 and VAPs in reticulophagy expands our understanding of this process.

Epr1 was first identified by Zhao et al. in Schizosaccharomyces pombe by exploiting affinity-purification coupled with mass spectrometry (AP-MS) analysis using Atg8 as bait. Through coimmunoprecipitation analysis and yeast two-hybrid assays, the authors confirmed that Epr1 interacts with Atg8 via an Atg8-family interacting motif (AIM) in its C-terminal disordered region. Using fluorescence microscopy, the authors found that the ER-localized protein Epr1 accumulates at the phagophore assembly site when bulk autophagy is triggered, and Epr1 is required for DTT-induced reticulophagy. Electron microscopy results suggest that Epr1 is important for the sequestration of ER membranes into phagophores under these conditions.

Through these methods, Zhao et al. found that Epr1 interacts with Scs2 and Scs22 through its FFAT motif. Scs2 and Scs22 are two ER-localized integral membrane proteins, which belong to the VAP family. Through checking its fluorescence localization and the utilization of a GST affinity-isolation assay, the authors demonstrated that the major role of Epr1 in reticulophagy is to mediate an Atg8-VAP association exploiting the AIM and FFAT motifs, which are located in the Epr1 C terminus.

Moreover, the authors demonstrated that binding Atg8 to a non-VAP ER membrane protein, Erg11, is able to rescue the reticulophagy defect of an epr1∆ knockout strain, but not the reticulophagy defect of an scs2∆ scs22∆ double knockout, suggesting additional roles of VAPs in mediating reticulophagy besides functioning as an ER anchor. Furthermore, by introducing an artificial ER-PM tether, the authors demonstrated that the role of VAPs in maintaining the ER-PM contact is essential for reticulophagy.

Zhao et al. then tested the growth ability of epr1∆ or scs2∆ scs22∆ cells to survive DTT treatment, and determined that VAP- and Epr1-mediated reticulophagy is crucial for surviving ER stress. In addition, the authors revealed that Epr1 is upregulated during ER stress probably translationally or post-translationally dependent on Ire1.

Taken together, the authors identified Epr1 as a novel reticulophagy receptor, which links Atg8 and VAPs through its C terminus AIM and FFAT motifs to confer selectivity. The authors also demonstrated that the ER-PM contact provided by VAPs as well as Ire1-dependent Epr1 upregulation are important for reticulophagy. Of note, another paper published in The EMBO Journal, indicated that another reticulophagy receptor, CALCOCO1, shares a similar mechanism in mammalian cells by interacting with Atg8-family proteins and VAPs to mediate reticulophagy [8], suggesting a conserved mechanism across species.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Institute of General Medical Sciences [GM131919].