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      Noncanonical E2 recruitment by the autophagy E1 revealed by Atg7–Atg3 and Atg7–Atg10 structures

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          Abstract

          Core functions of autophagy are mediated by ubiquitin-like protein (UBL) cascades, in which a homodimeric E1 enzyme, Atg7, directs the UBLs Atg8 and Atg12 to their respective E2 enzymes, Atg3 and Atg10. Crystallographic and mutational analyses of yeast (Atg7 – Atg3) 2 and (Atg7 –Atg10) 2 complexes reveal noncanonical, multisite E1 –E2 recognition in autophagy. Atg7’s unique N-terminal domain recruits distinctive elements from the Atg3 and Atg10 ‘backsides’. This, along with E1 and E2 conformational variability, allows presentation of ‘frontside’ Atg3 and Atg10 active sites to the catalytic cysteine in the C-terminal domain from the opposite Atg7 protomer in the homodimer. Despite different modes of binding, the data suggest that common principles underlie conjugation in both noncanonical and canonical UBL cascades, whereby flexibly tethered E1 domains recruit E2s through surfaces remote from their active sites to juxtapose the E1 and E2 catalytic cysteines.

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          Most cited references42

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          A ubiquitin-like system mediates protein lipidation.

          Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.
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            Dynamics and diversity in autophagy mechanisms: lessons from yeast.

            Autophagy is a fundamental function of eukaryotic cells and is well conserved from yeast to humans. The most remarkable feature of autophagy is the synthesis of double membrane-bound compartments that sequester materials to be degraded in lytic compartments, a process that seems to be mechanistically distinct from conventional membrane traffic. The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today. Analyses of autophagy-related (Atg) proteins have unveiled dynamic and diverse aspects of mechanisms that underlie membrane formation during autophagy.
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              A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

              Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.
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                Author and article information

                Journal
                101186374
                31761
                Nat Struct Mol Biol
                Nat. Struct. Mol. Biol.
                Nature structural & molecular biology
                1545-9993
                1545-9985
                2 October 2012
                11 November 2012
                December 2012
                01 June 2013
                : 19
                : 12
                : 1242-1249
                Affiliations
                [1 ]Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
                [2 ]Life Sciences Institute; University of Michigan; Ann Arbor, MI USA
                [3 ]Integrated Program in Biomedical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee, USA
                [4 ]Howard Hughes Medical Institute, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
                [5 ]Department of Chemistry and Chemical Biology, Cornell University, Argonne, IL 60439
                [6 ]Department of Bioengineering, Stanford University, Stanford, CA, USA
                Author notes
                Correspondence: Brenda A. Schulman, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS#311, Memphis, TN 38105, Phone: 901-595-5147, brenda.schulman@ 123456stjude.org
                Article
                NIHMS408910
                10.1038/nsmb.2415
                3515690
                23142976
                3e3238b7-9d9c-4e34-95f7-e8c00407ba96

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                History
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM053396 || GM
                Categories
                Article

                Molecular biology
                Molecular biology

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