Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop to promote autophagy

Abstract

Macroautophagy (autophagy) is driven by the coordinated actions of core autophagy-related (Atg) proteins. Atg8, the core Atg protein generally considered acting most downstream, has recently been shown to interact with other core Atg proteins via their Atg8-family-interacting motifs (AIMs). However, the extent, functional consequence, and evolutionary conservation of such interactions remain inadequately understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg38, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex I, interacts with Atg8 via an AIM, which is highly conserved in Atg38 proteins of fission yeast species, but not conserved in Atg38 proteins of other species. This interaction recruits Atg38 to Atg8 on the phagophore assembly site (PAS) and consequently enhances PAS accumulation of the PtdIns3K complex I and Atg proteins acting downstream of the PtdIns3K complex I, including Atg8. The disruption of the Atg38-Atg8 interaction leads to the reduction of autophagosome size and autophagic flux. Remarkably, the loss of this interaction can be compensated by an artificial Atg14-Atg8 interaction. Our findings demonstrate that the Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop between Atg8 and the PtdIns3K complex I to promote efficient autophagosome formation, underscore the prevalence and diversity of AIMmediated connections within the autophagic machinery, and reveal unforeseen flexibility of such connections.

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

“…By contrast, Atg11(522-583) rescued atg11Δ as well as full-length Atg11 ( Figure 1F ). Similar results were obtained using the Pho8Δ60 assay, where we expressed the autophagy reporter S. cerevisiae Pho8Δ60 in S. pombe pho8Δ background ( Yu et al, 2020 ; Figure 1G ). These results show that Atg11(522-583) is necessary and sufficient for the autophagy function of Atg11.…”

“…Pho8∆60 activity assay was performed as described in S. cerevisiae ( Noda et al, 1995 ) with some modifications ( Yu et al, 2020 ). About 5 OD 600 units of cells were collected and washed with 0.85% NaCl, and then resuspended in 200 μL of lysis buffer (20 mM PIPES, pH 6.8, 50 mM KCl, 100 mM KOAc, 10 mM MgSO 4 , 10 μM ZnSO 4 , 0.5% Triton X-100, 2 mM PMSF) and incubated at room temperature for 20 min.…”

A UPR-Induced Soluble ER-Phagy Receptor Acts with VAPs to Confer ER Stress Resistance

“…In recent years, the fission yeast Schizosaccharomyces pombe has become an important model organism for studying autophagy (Kohda et al, 2007;Liu et al, 2015;Mukaiyama et al, 2009Mukaiyama et al, , 2010Nanji et al, 2017;Sun et al, 2013;Suzuki et al, 2015;Yu et al, 2020;Zhao et al, 2016). In this work, we identified a previously uncharacterized S. pombe protein, Epr1, as an Atg8-interacting autophagy receptor required for ER stress-induced ER-phagy.…”

“…To test this idea, we examined whether Epr1 can be substituted by an artificial soluble tether that can interact simultaneously with both Atg8 and VAPs (Figure 4D). This tether consists of GFP, an artificial AIM (AIM art ) (Wu et al, 2015;Yu et al, 2020), and a (H) Quantification of the percentages of autophagosomes containing AuNP-labeled ER membranes observed in (G) (mean ± SD). Three independent experiments were performed.…”

Autophagy in Plant Abiotic Stress Management

“…Atg38 helps to maintain the integrity of the PtdIns3K complex. A previous study identified that Atg38 can also interact with Atg8 via an ATG8-interacting motif (AIM), which is responsible for the PAS accumulation of the PtdIns3K complex [ 31 ]. Sequence homologs of these genes, except ATG38, have been found in plants.…”