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The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D-CDC6 and D-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.

Autophagic receptor p62 is a critical mediator of cell detoxification, stress response, and metabolic programs and is commonly deregulated in human diseases. The diverse functions of p62 arise from its ability to interact with a large set of ligands, such as arginylated (Nt-R) substrates. Here, we describe the structural mechanism for selective recognition of Nt-R by the ZZ domain of p62 (p62ZZ). We show that binding of p62ZZ to Nt-R substrates stimulates p62 aggregation and macroautophagy and is required for autophagic targeting of p62. p62 is essential for mTORC1 activation in response to arginine, but it is not a direct sensor of free arginine in the mTORC1 pathway. We identified a regulatory linker (RL) region in p62 that binds p62ZZ in vitro and may modulate p62 function. Our findings shed new light on the mechanistic and functional significance of the major cytosolic adaptor protein p62 in two fundamental signaling pathways.

BiP and other endoplasmic reticulum (ER)-resident proteins are thought to be metabolically stable and to function primarily in the ER lumen. We sought to assess how the abundance of these proteins dynamically fluctuates in response to various stresses and how their subpopulations are relocated to non-ER compartments such as the cytosol. We showed that the molecular chaperone BiP (also known as GRP78) was short-lived under basal conditions and ER stress. The turnover of BiP was in part driven by its amino-terminal arginylation (Nt-arginylation) by the arginyltransferase ATE1, which generated an autophagic N-degron of the N-end rule pathway. ER stress elicited the formation of R-BiP, an effect that was increased when the proteasome was also inhibited. Nt-arginylation correlated with the cytosolic relocalization of BiP under the types of stress tested. The cytosolic relocalization of BiP did not require the functionality of the unfolded protein response or the Sec61- or Derlin1-containing translocon. A key inhibitor of the turnover and Nt-arginylation of BiP was HERP (homocysteine-responsive ER protein), a 43-kDa ER membrane-integrated protein that is an essential component of ER-associated protein degradation. Pharmacological inhibition of the ER-Golgi secretory pathway also suppressed R-BiP formation. Finally, we showed that cytosolic R-BiP induced by ER stress and proteasomal inhibition was routed to autophagic vacuoles and possibly additional metabolic fates. These results suggest that Nt-arginylation is a posttranslational modification that modulates the function, localization, and metabolic fate of ER-resident proteins.

The N-end rule pathway is a proteolytic system in which single N-terminal residues of proteins act as N-degrons. These degrons are recognized by N-recognins, facilitating substrate degradation via the ubiquitin (Ub) proteasome system (UPS) or autophagy. We have previously identified a set of N-recognins [UBR1, UBR2, UBR4 (also known as p600) and UBR5 (also known as EDD)] that bind N-degrons through their UBR boxes to promote proteolysis by the proteasome. Here, we show that the 570 kDa N-recognin UBR4 is associated with maturing endosomes through an interaction with Ca-bound calmodulin. The endosomal recruitment of UBR4 is essential for the biogenesis of early endosomes (EEs) and endosome-related processes, such as the trafficking of endocytosed protein cargos and degradation of extracellular cargos by endosomal hydrolases. In mouse embryos, UBR4 marks and plays a role in the endosome-lysosome pathway that mediates the heterophagic proteolysis of endocytosed maternal proteins into amino acids. By screening 9591 drugs through the DrugBank database, we identify picolinic acid as a putative ligand for UBR4 that inhibits the biogenesis of EEs. Our results suggest that UBR4 is an essential modulator in the endosome-lysosome system.This article has an associated First Person interview with the first author of the paper.

Although proteasome inhibitors (PIs) are used as anticancer drugs to treat various cancers, their relative therapeutic efficacy on stem cells vs. bulk cancers remains unknown. Here, we show that stem

No abstract

SQSTM1/p62 facilitates responses to various cellular stresses and has been implicated in human diseases. This protein functions as a major cytoplasmic signaling hub and has multiple binding partners, including arginylated (Nt-R) proteins that are recognized by the ZZ domain of SQSTM1/p62 (SQSTM1/p62 ZZ ). We have determined the molecular mechanism of Nt-R recognition using a combination of biochemical and NMR approaches and obtained the crystal structure of SQSTM1/p62 ZZ in complex with Nt-R. We found that binding of SQSTM1/p62 ZZ to Nt-R induces SQSTM1/p62 puncta formation and macroautophagy/autophagy and identified a regulatory linker (RL) region of SQSTM1/p62 that associates with SQSTM1/p62 ZZ in vitro. Our findings suggest a mechanism for SQSTM1/p62 autoregulation that can be essential in mediating autophagy. ARTICLE HISTORY