The autophagic degradation of cytosolic pools of peroxisomal proteins by a new selective pathway

Damaged or redundant peroxisomes and their luminal cargoes are removed by pexophagy, a selective autophagy pathway. In yeasts, pexophagy depends mostly on the pexophagy receptors, such as Atg30 for <i>Pichia pastoris</i> and Atg36 for <i>Saccharomyces cerevisia</i>e, the autophagy scaffold proteins, Atg11 and Atg17, and the core autophagy machinery. In <i>P. pastoris</i>, the receptors for peroxisomal matrix proteins containing peroxisomal targeting signals (PTSs) include the PTS1 receptor, Pex5, and the PTS2 receptor and co-receptor, Pex7 and Pex20, respectively. These shuttling receptors are predominantly cytosolic and only partially peroxisomal. It remains unresolved as to whether, when and how the cytosolic pools of peroxisomal receptors, as well as the peroxisomal matrix proteins, are degraded under pexophagy conditions. These cytosolic pools exist both in normal and mutant cells impaired in peroxisome biogenesis. We report here that Pex5 and Pex7, but not Pex20, are degraded by an Atg30-independent, selective autophagy pathway. To enter this selective autophagy pathway, Pex7 required its major PTS2 cargo, Pot1. Similarly, the degradation of Pex5 was inhibited in cells missing abundant PTS1 cargoes, such as alcohol oxidases and Fox2 (hydratase-dehydrogenase-epimerase). Furthermore, in cells deficient in PTS receptors, the cytosolic pools of peroxisomal matrix proteins, such as Pot1 and Fox2, were also removed by Atg30-independent, selective autophagy, under pexophagy conditions. In summary, the cytosolic pools of PTS receptors and their cargoes are degraded via a pexophagy-independent, selective autophagy pathway under pexophagy conditions. These autophagy pathways likely protect cells from futile enzymatic reactions that could potentially cause the accumulation of toxic cytosolic products. <b>Abbreviations:</b> ATG: autophagy related; Cvt: cytoplasm to vacuole targeting; Fox2: hydratase-dehydrogenase-epimerase; PAGE: polyacrylamide gel electrophoresis; Pot1: thiolase; PMP: peroxisomal membrane protein; Pgk1: 3-phosphoglycerate kinase; PTS: peroxisomal targeting signal; RADAR: receptor accumulation and degradation in the absence of recycling; RING: really interesting new gene; SDS: sodium dodecyl sulphate; TCA, trichloroacetic acid; Ub: ubiquitin; UPS: ubiquitin-proteasome system Vid: vacuole import and degradation

过氧化物酶体自噬（pexophagy）作为一种选择性自噬（selective autophagy）通路，负责清除受损或冗余的过氧化物酶体（peroxisome）及其腔内货物。在酵母中，过氧化物酶体自噬主要依赖于过氧化物酶体自噬受体，如毕赤酵母（Pichia pastoris）的Atg30与酿酒酵母（Saccharomyces cerevisiae）的Atg36、自噬支架蛋白Atg11和Atg17，以及核心自噬机制。

在毕赤酵母中，携带过氧化物酶体靶向信号（peroxisomal targeting signals, PTSs）的过氧化物酶体基质蛋白的受体包括PTS1受体Pex5，以及PTS2受体及其共受体，分别为Pex7和Pex20。这类穿梭受体主要定位于细胞质基质，仅少量定位于过氧化物酶体。目前仍不清楚，在过氧化物酶体自噬条件下，过氧化物酶体受体的细胞质池以及过氧化物酶体基质蛋白是否会被降解、何时被降解以及如何被降解。这类细胞质池既存在于正常细胞中，也存在于过氧化物酶体生物发生受损的突变细胞中。

本研究发现，Pex5与Pex7（而非Pex20）会通过不依赖Atg30的选择性自噬通路被降解。Pex7要进入该选择性自噬通路，需要其主要的PTS2货物蛋白Pot1。类似地，在缺乏丰富PTS1货物蛋白（如醇氧化酶与Fox2（水合酶-脱氢酶-表异构酶））的细胞中，Pex5的降解会受到抑制。此外，在过氧化物酶体自噬条件下，PTS受体缺陷细胞中的过氧化物酶体基质蛋白（如Pot1和Fox2）的细胞质池也会通过不依赖Atg30的选择性自噬通路被清除。

综上，在过氧化物酶体自噬条件下，PTS受体及其货物蛋白的细胞质池会通过不依赖过氧化物酶体自噬的选择性自噬通路被降解。这类自噬通路可能会保护细胞免受无效酶促反应的影响，而这类反应可能会导致毒性细胞质产物的积累。

**缩写说明：**
ATG（autophagy related）：自噬相关基因；
Cvt（cytoplasm to vacuole targeting）：细胞质至液泡靶向通路；
Fox2（hydratase-dehydrogenase-epimerase）：水合酶-脱氢酶-表异构酶；
PAGE（polyacrylamide gel electrophoresis）：聚丙烯酰胺凝胶电泳；
Pot1（thiolase）：硫解酶；
PMP（peroxisomal membrane protein）：过氧化物酶体膜蛋白；
Pgk1（3-phosphoglycerate kinase）：3-磷酸甘油酸激酶；
PTS（peroxisomal targeting signal）：过氧化物酶体靶向信号；
RADAR（receptor accumulation and degradation in the absence of recycling）：回收缺失时受体积累与降解通路；
RING（really interesting new gene）：环指结构域；
SDS（sodium dodecyl sulphate）：十二烷基硫酸钠；
TCA（trichloroacetic acid）：三氯乙酸；
Ub（ubiquitin）：泛素；
UPS（ubiquitin-proteasome system）：泛素-蛋白酶体系统；
Vid（vacuole import and degradation）：液泡输入与降解通路。