6B, compare lanes 2 with 3). 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 website of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable part 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 Nav1.7-IN-2 reporter through a distinct mechanism that bears notable similarity with the candida Cvt (cytoplasm-to-vacuole focusing on) pathway, and suggest the GST-BHMT reporter might be used like a easy assay to study selective macroautophagy in mammalian cells. led to the recognition of another group of novel parts required for the autophagy-dependent degradation of P-granules.8 Notably, in both the Cvt and P-granule pathways, sequestration of cargos into autophagosomes is likely ubiquitin-independent,7,9 whereas in the mammalian system, cargos that are sequestered from the selective pathway often consist of specific modifications such as ubiquitination.10 In particular, selective autophagy often requires the presence of receptor proteins such as SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1), the mammalian ortholog of yeast Atg19, which contains both a ubiquitin binding website and a MAP1LC3 (microtubule-associated protein 1 light chain 3)-interacting motif to bridge the sequestration of ubiquitin-modified cargos into the autophagosome.11 Another important part of the autophagic response is to keep up intracellular quality control and counteract cellular stress.12 The autophagy-lysosome pathway works together with the ubiquitin-proteasome system (UPS), another cellular clearance mechanism, to degrade misfolded or unwanted proteins. In agreement with the important roles of these pathways in conserving protein homeostasis (proteostasis) in the cell, dysfunction in both pathways has been linked to irregular build up of ubiquitinated protein aggregates in the cell. For example, inactivating basal levels of cellular autophagy by depleting ATG5 (autophagy-related 5) or ATG7 in mouse mind leads to protein aggregation and neurodegeneration.13,14 Similarly, disruption of proteasomal function also results in the accumulation of abnormal protein aggregates.15 Available evidence supports the existence of intercommunication between these 2 important cellular protective mechanisms.16 For example, software of the chemical compound MG132, a Nav1.7-IN-2 specific and reversible proteasome inhibitor, can induce autophagy.17,18 It is assumed that this MG132-induced autophagic activation is an indirect cellular compensatory response, possibly mediated by ER (endoplasmic reticulum) pressure or MAPK11/12/13/14 (mitogen-activated protein kinase 11/12/13/14) signaling pathways, to offset jeopardized proteasomal activity and maintain proper proteostasis.17,19 However, the detailed mechanism of this MG132-induced autophagic activation is still unclear. The GST-BHMT (a fusion protein of GST [glutathionine S-transferase] tagged to the N terminus of BHMT [betaine-homocysteine S-methyltransferase]) reporter has recently been developed as an endpoint, cargo-based assay for the study of autophagy.20,21 The endogenous BHMT enzyme is highly indicated in liver and kidney cells. BHMT like a cargo is definitely delivered through the autophagy pathway into the lysosome where it is cleaved at its N-terminal loop site by asparaginyl endopeptidase LGMN (legumain) to produce a specific proteolytic fragment (BHMT(FRAG)).22 Further, this specific cleavage event responds to amino acid IL22RA2 or serum starvation in an Nav1.7-IN-2 autophagy-dependent manner. Accordingly, the build up of a GST-tagged version of the cleaved BHMT product (GST-BHMT(FRAG)) has been successfully used like a cargo-based, endpoint reporter to monitor starvation-induced autophagy activity in different cell lines.21 However, whether the GST-BHMT assay is applicable to.