Person: Kamber, Roarke Alexander
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Kamber
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Roarke Alexander
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Kamber, Roarke Alexander
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Publication Mechanisms Regulating Target Selection and Degradation by Selective Autophagy(2016-09-12) Kamber, Roarke Alexander; Bernhardt, Thomas; Finley, Daniel; Walther, Tobias; Laub, MichaelSelective autophagy eliminates protein aggregates, damaged organelles, and other targets that otherwise accumulate and cause disease. A class of proteins called autophagy receptors is known to select targets for degradation, but it has remained unclear 1) whether target recognition by autophagy receptors is mechanistically coupled with autophagy initiation and 2) how autophagy receptors select individual organelle targets. We found that autophagy receptors activate the autophagy master kinase, Atg1, upon target detection. Specifically, we found that in nutrient-rich conditions, Atg1 is active only in a multisubunit complex comprising constitutive protein aggregates, their autophagy receptor, and a scaffold protein, Atg11. Development of a cell-free assay for Atg1-mediated phosphorylation enabled us to activate Atg1 with purified receptor-bound aggregates and Atg11. Another target, damaged peroxisomes, also activated Atg1 using Atg11 with a distinct receptor, Atg36. This work revealed a key piece of signaling logic: receptor-bound targets drive selective autophagy locally by activating Atg1, thereby coupling the rate of autophagy initiation with the rate of target appearance. To understand how autophagy receptors are themselves regulated by phosphorylation to enable selection of individual targets, we studied Atg36 phosphoactivation by the cytosolic kinase Hrr25, which is known to drive Atg36-Atg11 binding. Biochemical isolation of Atg36 in complex with its peroxisome anchor protein, Pex3, revealed that Pex3 and Atg36 exist in a multisubunit complex with the peroxisomal Pex1/6/15 AAA-ATPase complex, which had previously been shown to extract ubiquitinated Pex5 from the peroxisome membrane. We found that Pex5 is not involved in Atg36 regulation, whereas loss of Pex1/6/15 causes Atg36 to become constitutively active, suggesting the peroxisomal AAA-ATPase complex has a second function in pexophagy regulation. By retargeting these proteins to an orthologous cellular location, the mitochondrial outer membrane, we showed that Pex1/6/15 are sufficient to inhibit Atg36 independent of other peroxisome proteins. This work uncovered a novel mechanism by which an autophagy receptor is regulated by target-localized factors to enable organelle-specific autophagy decisions.Publication Receptor-Bound Targets of Selective Autophagy Use a Scaffold Protein to Activate the Atg1 Kinase(Elsevier BV, 2015) Kamber, Roarke Alexander; Shoemaker, Christopher; Denic, VladimirSelective autophagy eliminates protein aggregates, damaged organelles, and other targets that otherwise accumulate and cause disease. Autophagy receptors mediate selectivity by connecting targets to the autophagosome membrane. It has remained unknown whether receptors perform additional functions. Here, we show that in yeast certain receptor-bound targets activate Atg1, the kinase that controls autophagosome formation. Specifically, we found that in nutrient-rich conditions, Atg1 is active only in a multi-subunit complex comprising constitutive protein aggregates, their autophagy receptor, and a scaffold protein Atg11. Development of a cell-free assay for Atg1-mediated phosphorylation enabled us to activate Atg1 with purified receptorbound aggregates and Atg11. Another target, damaged peroxisomes, also activated Atg1 using Atg11 with a distinct receptor. Our work reveals that receptor-target complexes activate Atg1 to drive formation of selective autophagosomes. This regulatory logic is a key similarity between selective autophagy and bulk autophagy, which is initiated by a distinct Atg1 activation mechanism during starvation.