The selective macroautophagic degradation of aggregated proteins requires the PI3P-binding protein Alfy.

There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation and can occur selectively for substrates, including aggregated proteins. It remains unclear, however, whether starvation-induced and selective macroautophagy share identical adaptor molecules to capture their cargo. Here, we report that Alfy, a phosphatidylinositol 3-phosphate-binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L, and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner and, likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy by bridging cargo to the molecular machinery that builds autophagosomes.

[1]  Eeva-Liisa Eskelinen,et al.  3D tomography reveals connections between the phagophore and endoplasmic reticulum , 2009, Autophagy.

[2]  Ivan Dikic,et al.  Nix is a selective autophagy receptor for mitochondrial clearance , 2010, EMBO reports.

[3]  A. Isaacs,et al.  Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease , 2007, The Journal of cell biology.

[4]  Kostas Vekrellis,et al.  Wild Type α-Synuclein Is Degraded by Chaperone-mediated Autophagy and Macroautophagy in Neuronal Cells* , 2008, Journal of Biological Chemistry.

[5]  M. Komatsu,et al.  A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. , 2009, Molecular cell.

[6]  A. Brech,et al.  Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila , 2008, Autophagy.

[7]  R. Nixon,et al.  Neuronal macroautophagy: from development to degeneration. , 2006, Molecular aspects of medicine.

[8]  T. P. Neufeld,et al.  Role and regulation of starvation-induced autophagy in the Drosophila fat body. , 2004, Developmental cell.

[9]  H. Kampinga,et al.  Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy. , 2002, Human molecular genetics.

[10]  John L Cleveland,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes , 2008, Autophagy.

[11]  H. Sandoval,et al.  Essential role for Nix in autophagic maturation of erythroid cells , 2008, Nature.

[12]  Harry T Orr,et al.  Recovery from Polyglutamine-Induced Neurodegeneration in Conditional SCA1 Transgenic Mice , 2004, The Journal of Neuroscience.

[13]  S. Benzer,et al.  Genetic suppression of polyglutamine toxicity in Drosophila. , 2000, Science.

[14]  A. Brech,et al.  A dual function for Deep orange in programmed autophagy in the Drosophila melanogaster fat body. , 2006, Experimental cell research.

[15]  S. Schreiber,et al.  Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. , 2007, Nature chemical biology.

[16]  Janghoo Lim,et al.  ATAXIN-1 Interacts with the Repressor Capicua in Its Native Complex to Cause SCA1 Neuropathology , 2006, Cell.

[17]  T. Noda,et al.  The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. , 2008, Molecular biology of the cell.

[18]  S. Bloor,et al.  The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria , 2009, Nature Immunology.

[19]  René Hen,et al.  Reversal of Neuropathology and Motor Dysfunction in a Conditional Model of Huntington's Disease , 2000, Cell.

[20]  M. MacDonald,et al.  Huntingtin's WW domain partners in Huntington's disease post-mortem brain fulfill genetic criteria for direct involvement in Huntington's disease pathogenesis. , 2000, Human molecular genetics.

[21]  Terje Johansen,et al.  p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death , 2005, The Journal of cell biology.

[22]  Fumiaki Tanaka,et al.  Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. , 2003, Human molecular genetics.

[23]  Susan Cheng,et al.  Genetic Modifiers of the Drosophila Blue Cheese Gene Link Defects in Lysosomal Transport With Decreased Life Span and Altered Ubiquitinated-Protein Profiles , 2007, Genetics.

[24]  Leonidas Stefanis,et al.  Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. , 2004, Science.

[25]  Takeshi Noda,et al.  LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing , 2000, The EMBO journal.

[26]  S. Tooze,et al.  Microtubules Facilitate Autophagosome Formation and Fusion of Autophagosomes with Endosomes , 2006, Traffic.

[27]  H. Lehrach,et al.  Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates. , 1999, Methods in enzymology.

[28]  Harald Stenmark,et al.  Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes , 2004, Journal of Cell Science.

[29]  J. Vance,et al.  The Deacetylase HDAC6 Regulates Aggresome Formation and Cell Viability in Response to Misfolded Protein Stress , 2003, Cell.

[30]  H. Glaumann,et al.  Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization , 1982, The Journal of cell biology.

[31]  Rainer Duden,et al.  Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. , 2002, Human molecular genetics.

[32]  Francesco Scaravilli,et al.  Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease , 2004, Nature Genetics.

[33]  D. Klionsky,et al.  Atg32 is a mitochondrial protein that confers selectivity during mitophagy. , 2009, Developmental cell.

[34]  R. Kopito,et al.  Aggresomes: A Cellular Response to Misfolded Proteins , 1998, The Journal of cell biology.

[35]  J. Rothman,et al.  triggered by the insulin-signaling pathway , 2006 .

[36]  Nancy M Bonini,et al.  Drosophila as a model for human neurodegenerative disease. , 2005, Annual review of genetics.

[37]  A. von Mikecz,et al.  Proteasomes degrade proteins in focal subdomains of the human cell nucleus , 2005, Journal of Cell Science.

[38]  J. Rothman,et al.  Autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway , 2006, The Journal of cell biology.

[39]  S. W. Davies,et al.  Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. , 1997, Science.

[40]  P. Seglen,et al.  Purification and characterization of autophagosomes from rat hepatocytes. , 1998, The Biochemical journal.

[41]  T. Natsume,et al.  Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate , 2003, Journal of Cell Science.

[42]  D. Housman,et al.  Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila , 2001, Nature.

[43]  Mark R. Segal,et al.  Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death , 2004, Nature.

[44]  R. Kopito,et al.  HDAC6 and Microtubules Are Required for Autophagic Degradation of Aggregated Huntingtin* , 2005, Journal of Biological Chemistry.

[45]  C. Ross,et al.  Polyglutamine expansion of huntingtin impairs its nuclear export , 2005, Nature Genetics.

[46]  T. Noda,et al.  A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation , 2009, Nature Cell Biology.

[47]  D. Klionsky,et al.  Molecular Mechanisms and Regulation of Specific and Nonspecific Autophagy Pathways in Yeast* , 2005, Journal of Biological Chemistry.

[48]  P. Seglen,et al.  Isolation and Characterization of Rat Liver Amphisomes , 1998, The Journal of Biological Chemistry.

[49]  Dan Garza,et al.  HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS , 2007, Nature.

[50]  P. Seglen,et al.  Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. , 2004, Developmental cell.

[51]  Y. Ohsumi,et al.  Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy. , 2009, Developmental cell.

[52]  Gareth Griffiths,et al.  Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum , 2008, The Journal of cell biology.

[53]  M. Mckeown,et al.  blue cheese Mutations Define a Novel, Conserved Gene Involved in Progressive Neural Degeneration , 2003, The Journal of Neuroscience.

[54]  Peter T. Lansbury,et al.  Impaired Degradation of Mutant α-Synuclein by Chaperone-Mediated Autophagy , 2004, Science.

[55]  Atsushi Iwata,et al.  Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[56]  G. Bjørkøy,et al.  p62/SQSTM1 Binds Directly to Atg8/LC3 to Facilitate Degradation of Ubiquitinated Protein Aggregates by Autophagy* , 2007, Journal of Biological Chemistry.

[57]  Mark Turmaine,et al.  Formation of Neuronal Intranuclear Inclusions Underlies the Neurological Dysfunction in Mice Transgenic for the HD Mutation , 1997, Cell.

[58]  N. Mizushima,et al.  Generation of cell lines with tetracycline‐regulated autophagy and a role for autophagy in controlling cell size , 2006, FEBS letters.

[59]  F. Inagaki,et al.  The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy* , 2007, Journal of Biological Chemistry.