Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy

Abstract

Ribosomes are abundant cellular machines1,2 regulated by assembly, supernumerary subunit turnover, and nascent chain quality control mechanisms1–5. Moreover, nitrogen starvation in yeast has been reported to promote selective ribosome delivery to the vacuole in an autophagy conjugation system-dependent manner, a process called “ribophagy”6,7. However, whether ribophagy in mammals is selective or regulated is unclear. Using Ribo-Keima flux reporters, we find that starvation or mTOR inhibition promotes VPS34-dependent ribophagic flux, which unlike yeast, is largely ATG8 conjugation independent and occurs concomitantly with other cytosolic protein autophagic flux reporters8,9. Ribophagic flux was not induced upon inhibition of translational elongation or nascent chain uncoupling, but was induced in a comparatively selective manner upon proteotoxic stress via arsenite10 or chromosome mis-segregation11 dependent upon VPS34 and ATG8 conjugation. Unexpectedly, agents typically used to induce selective autophagy also promoted increased ribosome and cytosolic protein reporter flux, suggesting significant bulk or “by-stander” autophagy during what is often considered selective autophagy12,13. These results emphasize the importance of monitoring non-specific cargo flux when assessing selective autophagy pathways.

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNARE s

“…The HEK293 STX16/ STX17 DKO cells showed significantly fewer autolysosomal puncta originating from RPS3-Keima than in parental WT cells (Fig 4C and D). Thus, the use of previously characterized Keima ribophagy probes (An & Harper, 2018) further confirmed the importance of having both Stx16 and Stx17 for autophagic cargos to arrive into acidified compartments, including during ribophagy. We generated STX16/STX17 DKO in these cells as well using CRISPR/Cas9 ( Fig 4B).…”

“…In functional autolysosomes, despite being in a degradative compartment, Keima protein is stable but changes its spectral properties (illustrated in Fig 4A) from maximum excitation wavelength at 440 nm in neutral pH to 586 nm in acidic pH, with emission remaining constant at 620 nm (Violot et al, 2009). We applied Keima to monitor ribophagy (An & Harper, 2018) and generated STX16/STX17 DKO using CRISPR/Cas9 ( Fig 4B) in HEK293 cells stably expressing RPS3-Keima (fusion between 40S ribosomal protein S3 and mKeima) protein (An & Harper, 2018). We applied Keima to monitor ribophagy (An & Harper, 2018) and generated STX16/STX17 DKO using CRISPR/Cas9 ( Fig 4B) in HEK293 cells stably expressing RPS3-Keima (fusion between 40S ribosomal protein S3 and mKeima) protein (An & Harper, 2018).…”

“…These shifts can be monitored using 440-nm and 560-nm excitation filters and emission at 620 nm in an HCM Cellomics system (see Materials and Methods). Under starvation conditions, we examined ribophagy as previously reported (An & Harper, 2018), by monitoring progression of RPS3-Keima into acidic autolysosomal compartments via quantification of cytoplasmic puncta at 560 nm excitation and 620 emission wavelengths (Fig 4C and D). RPS3-Keima was diffuse cytoplasmic in untreated cells.…”

“…Autophagy can be bulk or selective (Birgisdottir et al, 2013;Randow & Youle, 2014;Stolz et al, 2014), whereas the multiple biological outputs of autophagy depend on its completion and the type of termination of the autophagic pathway, e.g., degradation or secretion (Ponpuak et al, 2015;Levine & Kroemer, 2019). Bulk autophagy, whereby portions of the cytoplasm of heterogeneous composition are sequestered, occurs during starvation and can be quantified by imaging (An & Harper, 2018) or biochemically (Seglen et al, 2015;Engedal & Seglen, 2016) by following sequestration of the cytosolic enzyme LDH, or ultrastructurally by enumerating partially degraded electron-dense ribosomes (Tanaka et al, 2000;Eskelinen, 2008). Autophagy can be selectively guided to specific cargo via a number of sequestosome 1/p62-like receptors (SLRs; Bjorkoy et al, 2005;Birgisdottir et al, 2013;Stolz et al, 2014) or other classes of receptors besides SLRs (Kimura et al, 2016;Levine & Kroemer, 2019).…”

“…Examples of selective degradative autophagy include autophagy of mitochondria (mitophagy), peroxisomes (pexophagy), intracellular microbes (xenophagy), ribosomes (ribophagy), protein aggregates (aggrephagy), and specific intracellular multiprotein complexes (precision autophagy) (Birgisdottir et al, 2013;Randow & Youle, 2014;Kimura et al, 2016;An & Harper, 2018). Examples of selective degradative autophagy include autophagy of mitochondria (mitophagy), peroxisomes (pexophagy), intracellular microbes (xenophagy), ribosomes (ribophagy), protein aggregates (aggrephagy), and specific intracellular multiprotein complexes (precision autophagy) (Birgisdottir et al, 2013;Randow & Youle, 2014;Kimura et al, 2016;An & Harper, 2018).…”