The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells.
By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either ... pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.
Mesh Terms:
AMP-Activated Protein Kinases, Adaptor Proteins, Signal Transducing, Apoptosis Regulatory Proteins, Autophagy, Betaine-Homocysteine S-Methyltransferase, Endoplasmic Reticulum Stress, Endoribonucleases, Glutathione Transferase, HEK293 Cells, HeLa Cells, Humans, Leupeptins, Lysosomes, Membrane Proteins, Mitogen-Activated Protein Kinase 8, Proteasome Endopeptidase Complex, Proteasome Inhibitors, Protein Binding, Protein Multimerization, Protein Processing, Post-Translational, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Proteins, Recombinant Fusion Proteins, Signal Transduction, TOR Serine-Threonine Kinases, Ubiquitination
AMP-Activated Protein Kinases, Adaptor Proteins, Signal Transducing, Apoptosis Regulatory Proteins, Autophagy, Betaine-Homocysteine S-Methyltransferase, Endoplasmic Reticulum Stress, Endoribonucleases, Glutathione Transferase, HEK293 Cells, HeLa Cells, Humans, Leupeptins, Lysosomes, Membrane Proteins, Mitogen-Activated Protein Kinase 8, Proteasome Endopeptidase Complex, Proteasome Inhibitors, Protein Binding, Protein Multimerization, Protein Processing, Post-Translational, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Proteins, Recombinant Fusion Proteins, Signal Transduction, TOR Serine-Threonine Kinases, Ubiquitination
Autophagy
Date: May. 20, 2015
PubMed ID: 25984893
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