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.
Keywords: ACACA/B, acetyl-CoA carboxylase α/β; ACTB, actin, β; ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; ATG7, autophagy-related 7; BCL2, B-cell CLL/lymphoma 2; BECN1, Beclin 1, autophagy-related; BHMT; BHMT, betaine-homocysteine S-methyltransferase; Baf A1, bafilomycin A1; CTNNB1, catenin (cadherin-associated protein), β 1, 88kDa; Cvt, cytoplasm-to-vacuole-targeting; DDIT3, DNA-damage-inducible transcript 3; EBSS, Earle's Balanced Salt Solution; EIF2AK3, eukaryotic translation initiation factor 2-α, kinase 3; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; ER, endoplasmic reticulum; ERN1, endoplasmic reticulum to nucleus signaling 1; GST, glutathionine S-transferase; GST-BHMT(FRAG), an autophagy-mediated cleavage product of the GST-BHMT reporter; GST-BHMT, a fusion protein of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase; HA, hemagglutinin; HSPA5, heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa); LSCS, linker-specific cleavage site; MAP1LC3, microtubule-associated protein 1 light chain 3; MAP2K7, mitogen-activated protein kinase kinase 7; MAPK8, mitogen-activated protein kinase 8; MTOR; MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC1, MTOR complex 1; NBR1, neighbor of BRCA1 gene 1; P4HB, prolyl 4-hydroxylase, β polypeptide; PRKAA, protein kinase, AMP-activated, α catalytic subunit; PRKAA/AMPK; RHEB, Ras homolog enriched in brain; RM, rich medium; RPS6KB1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; SQSTM1, sequestosome 1; TSC1/2, tuberous sclerosis 1/2; ULK1, unc-51 like autophagy activating kinase 1; UPR, unfolded protein response; UPS, ubiquitin proteasome system; XBP1, X-box binding protein 1; cargo receptors SQSTM1/p62 and NBR1; proteasome inhibition; selective macroautophagy.