Autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as non-selective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-relevant proteins covering process-relevant proteins such as the initiation complex and the ATG8 lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy inducing conditions in contrast to SARs. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy fluxes.

These are the experiments linked to the publication talking about the monitoring of autophagy fluxes by PRM. PA14 Are calibration curves PA15 are starvation experiments performed in A549 cells PA12B are iron chelation experiments performed in AREP19 cells PA16 are methods comparison experiments

Samples were cells from mammalian cell culture. Either A549 lung carcinoma or AREP19 retinal cells expression the Mito-QC reporter. Samples were spiked with heavy-labelled synthetic peptides.