Cancer Research UK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Crewe Road South, Edinburgh, UK.
Institute for Biochemistry and Molecular Biology, Medical Faculty, University of Bonn, Bonn, Germany.
Nat Commun. 2024 Sep 5;15(1):7743. doi: 10.1038/s41467-024-52049-3.
Autophagy is a finely orchestrated process required for the lysosomal degradation of cytosolic components. The final degradation step is essential for clearing autophagic cargo and recycling macromolecules. Using a CRISPR/Cas9-based screen, we identify RNAseK, a highly conserved transmembrane protein, as a regulator of autophagosome degradation. Analyses of RNAseK knockout cells reveal that, while autophagosome maturation is intact, cargo degradation is severely disrupted. Importantly, lysosomal protease activity and acidification remain intact in the absence of RNAseK suggesting a specificity to autolysosome degradation. Analyses of lysosome fractions show reduced levels of a subset of hydrolases in the absence of RNAseK. Of these, the knockdown of PLD3 leads to a defect in autophagosome clearance. Furthermore, the lysosomal fraction of RNAseK-depleted cells exhibits an accumulation of the ESCRT-III complex component, VPS4a, which is required for the lysosomal targeting of PLD3. Altogether, here we identify a lysosomal hydrolase delivery pathway required for efficient autolysosome degradation.
自噬是一个需要溶酶体降解胞质成分的精细协调过程。最终的降解步骤对于清除自噬货物和回收大分子是必不可少的。使用基于 CRISPR/Cas9 的筛选，我们确定 RNAseK，一种高度保守的跨膜蛋白，是自噬体降解的调节剂。对 RNAseK 敲除细胞的分析表明，虽然自噬体成熟完整，但货物降解严重受损。重要的是，在没有 RNAseK 的情况下，溶酶体蛋白酶活性和酸化仍然完整，这表明其具有特异性降解自溶酶体。对溶酶体部分的分析表明，在没有 RNAseK 的情况下，一组水解酶的水平降低。在这些水解酶中，PLD3 的敲低导致自噬体清除缺陷。此外，RNAseK 耗尽细胞的溶酶体部分表现出 ESCRT-III 复合物成分 VPS4a 的积累，VPS4a 是 PLD3 溶酶体靶向所必需的。总之，我们在这里确定了一个溶酶体水解酶递呈途径，该途径对于有效的自溶酶体降解是必需的。
