In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.

中文翻译：

---

Pex3 通过调节 Hrr25 介导的磷酸化和蛋白酶体降解将 Atg36 的 pexophagy 受体活性限制在过氧化物酶体上。

在巨自噬（以下简称自噬）中，细胞质分子和细胞器随机或选择性地隔离在称为自噬体的双膜囊泡中，并被输送到溶酶体或液泡进行降解。在选择性自噬中，降解目标的特异性由自噬受体决定。在出芽的酵母酿酒酵母中，自噬受体与特定靶标和 Atg11 相互作用，导致蛋白质复合物的募集，从而启动自噬体的形成。先前的研究表明，自噬受体受翻译后修饰的调节。在过氧化物酶体的选择性自噬 (pexophagy) 中，受体 Atg36 通过与过氧化物酶体膜蛋白 Pex3 结合而定位于过氧化物酶体。我们之前报道过 Atg36 被 Hrr25（酪蛋白激酶 1δ）磷酸化，增加了 Atg36-Atg11 的相互作用，从而刺激了外食性开始。然而，Atg36 磷酸化的调控机制尚不清楚。在这里，我们表明 Atg36 磷酸化在缺乏 Pex3 或表达与 Atg36 相互作用有缺陷的 Pex3 突变体中被消除，这表明与 Pex3 的相互作用对于 Hrr25 介导的 Atg36 磷酸化是必不可少的。使用重组蛋白，我们进一步证明 Pex3 直接促进 Hrr25 对 Atg36 的磷酸化。共免疫沉淀分析表明 Atg36 与 Hrr25 的相互作用取决于 Pex3。这些结果表明 Pex3 增加了 Atg36-Hrr25 相互作用，从而刺激了过氧化物酶体膜上的 Atg36 磷酸化。此外，我们发现 Pex3 结合保护 Atg36 免受蛋白酶体降解。因此，Pex3 通过增强过氧化物酶体在该细胞器上的磷酸化和稳定性，将 Atg36 的活性限制在过氧化物酶体上。