The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast.

Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.

The hallmark domain of the oldest autophagy receptor family is a cargo-binding module.

In selective macroautophagy/autophagy, autophagy receptors are key molecules that determine cargo specificity. Most known autophagy receptors only exist in some but not all eukaryotic lineages. The exception is Nbr1 proteins, which are conserved across eukaryotes. The four-tryptophan (FW) domain is the hallmark of Nbr1 proteins, but its function has been unknown. Our recent study found that the FW domain in the Nbr1 protein of the filamentous fungus Chaetomium thermophilum binds the α-mannosidase Ams1, a known selective autophagy cargo in budding yeast and fission yeast. Furthermore, we showed that when C. thermophilum Nbr1 and Ams1 are expressed heterologously in fission yeast, FW domain-mediated binding can promote autophagic delivery of Ams1 into vacuoles. We solved the structure of the FW-Ams1 complex and revealed the structural mechanism underlying Ams1 recognition by the FW domain. The N-terminal di-glycine peptide of Ams1 fits into a conserved pocket of the FW domain. We propose that this cargo-binding mechanism may also be employed by Nbr1 proteins in other eukaryotes.

Structural mechanism of protein recognition by the FW domain of autophagy receptor Nbr1.

Neighbor of BRCA1 (Nbr1) is a conserved autophagy receptor that provides cargo selectivity to autophagy. The four-tryptophan (FW) domain is a signature domain of Nbr1, but its exact function remains unclear. Here, we show that Nbr1 from the filamentous fungus Chaetomium thermophilum uses its FW domain to bind the α-mannosidase Ams1, a cargo of selective autophagy in both budding yeast and fission yeast, and delivers Ams1 to the vacuole by conventional autophagy in heterologous fission yeast. The structure of the Ams1-FW complex was determined at 2.2 Å resolution by cryo-electron microscopy. The FW domain adopts an immunoglobulin-like ß-sandwich structure and recognizes the quaternary structure of the Ams1 tetramer. Notably, the N-terminal di-glycine of Ams1 is specifically recognized by a conserved pocket of the FW domain. The FW domain becomes degenerated in fission yeast Nbr1, which binds Ams1 with a ZZ domain instead. Our findings illustrate the protein binding mode of the FW domain and reveal the versatility of Nbr1-mediated cargo recognition.

Atg11-mediated activation of Atg1 kinase in fission yeast.

The protein kinase Atg1 is a key player in macroautophagy/autophagy, but how its activity is regulated in various organisms is inadequately understood. Our recent study showed that in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity depends on Atg11, but not Atg13, Atg17, or Atg101. Notably, a 62 amino acid region of S. pombe Atg11 is sufficient for activating Atg1. This region is composed of two parts: an Atg1-binding domain and a homodimerization domain. Atg11 uses this region to dimerize Atg1. Dimerized Atg1 is activated through cis-autophosphorylation.

Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation.

Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity. This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.