title: Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation.
abstract: Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.
authors.affiliation: Hospital for Sick Children, Toronto, ON M5G 0A4, Canada Biochemistry Department, University of Toronto, Toronto ON M5S 1A1, Canada.
authors.forename: Frozan
authors.initials: F
authors.lastname: Safi
authors.affiliation: Hospital for Sick Children, Toronto, ON M5G 0A4, Canada Biochemistry Department, University of Toronto, Toronto ON M5S 1A1, Canada.
authors.forename: Alina
authors.initials: A
authors.lastname: Shteiman-Kotler
authors.affiliation: Hospital for Sick Children, Toronto, ON M5G 0A4, Canada Biochemistry Department, University of Toronto, Toronto ON M5S 1A1, Canada.
authors.forename: Yunan
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Iliadi
authors.affiliation: Hospital for Sick Children, Toronto, ON M5G 0A4, Canada Molecular Genetics Department, University of Toronto, Toronto ON M5S 1A1, Canada.
authors.forename: Gabrielle L
authors.initials: GL
authors.lastname: Boulianne
authors.affiliation: Hospital for Sick Children, Toronto, ON M5G 0A4, Canada Biochemistry Department, University of Toronto, Toronto ON M5S 1A1, Canada drotin@sickkids.ca.
authors.forename: Daniela
authors.initials: D
authors.lastname: Rotin
mesh_terms: D000818:Animals; D001665:Binding Sites; D015536:Down-Regulation; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000254:growth & development / Q000378:metabolism*; D007814:Larva / Q000254:growth & development / Q000378:metabolism; D024510:Muscle Development; D009132:Muscles / Q000378:metabolism* / Q000473:pathology; D000075702:Nedd4 Ubiquitin Protein Ligases; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C075637:amphiphysin; D000075702:Nedd4 Ubiquitin Protein Ligases; C000605755:Nedd4 protein, Drosophila; D044767:Ubiquitin-Protein Ligases
references.citation: Development. 1990 Nov;110(3):791-804
references.pmid: 2100994
references.citation: Curr Opin Neurobiol. 2007 Feb;17(1):35-42
references.pmid: 17229568
references.citation: PLoS Biol. 2010 Feb;8(2):e1000312
references.pmid: 20169111
references.citation: J Biol Chem. 2004 Apr 16;279(16):16017-25
references.pmid: 14761940
references.citation: J Biol Chem. 1997 May 16;272(20):13419-25
references.pmid: 9148966
references.citation: Traffic. 2008 Dec;9(12):2190-205
references.pmid: 18796008
references.citation: Traffic. 2001 Nov;2(11):839-50
references.pmid: 11733051
references.citation: Neuron. 2000 May;26(2):313-29
references.pmid: 10839352
references.citation: J Physiol. 2013 Jul 1;591(13):3133-43
references.pmid: 23613532
references.citation: Trends Genet. 1999 Nov;15(11):448-53
references.pmid: 10529807
references.citation: Neuron. 2002 Feb 28;33(5):789-804
references.pmid: 11879655
references.citation: Curr Biol. 1999 Jun 3;9(11):589-92
references.pmid: 10359699
references.citation: Development. 2001 Dec;128(24):5005-15
references.pmid: 11748137
references.citation: J Vis Exp. 2009;(24). pii: 1107. doi: 10.3791/1107
references.pmid: 19229190
references.citation: Mol Cell Biol. 2003 Jun;23(12):4295-306
references.pmid: 12773571
references.citation: Genes Dev. 2001 Nov 15;15(22):2967-79
references.pmid: 11711432
references.citation: EMBO J. 1992 Jul;11(7):2521-30
references.pmid: 1628617
references.citation: Cell. 1993 Jul 16;74(1):83-91
references.pmid: 8334708
references.citation: Nat Med. 2014 Jun;20(6):624-32
references.pmid: 24836577
references.citation: PLoS One. 2011;6(11):e27007
references.pmid: 22110599
references.citation: Cell. 1998 Jun 12;93(6):921-7
references.pmid: 9635422
references.citation: Mol Cell Biol. 2007 Jan;27(2):481-96
references.pmid: 17074801
references.citation: Science. 2002 Aug 16;297(5584):1193-6
references.pmid: 12183633
references.citation: FEBS J. 2010 Jul;277(13):2803-14
references.pmid: 20491914
references.citation: Nat Rev Mol Cell Biol. 2009 Jun;10(6):398-409
references.pmid: 19436320
references.citation: Front Physiol. 2013 Dec 12;4:363
references.pmid: 24376424
references.citation: Mol Biol Cell. 2013 May;24(9):1444-53
references.pmid: 23447705
references.citation: Wiley Interdiscip Rev Dev Biol. 2015 Jul-Aug;4(4):357-75
references.pmid: 25728002
references.citation: PLoS One. 2011;6(9):e25740
references.pmid: 21984944
references.citation: Development. 1998 Oct;125(19):3853-63
references.pmid: 9729493
references.citation: Physiol Rev. 2002 Apr;82(2):373-428
references.pmid: 11917093
references.citation: Circ Res. 2014 Dec 5;115(12):986-96
references.pmid: 25332206
references.citation: Skelet Muscle. 2011 Jul 13;1(1):26
references.pmid: 21797990
references.citation: Science. 2004 Jan 23;303(5657):495-9
references.pmid: 14645856
references.citation: Nature. 2001 Jul 26;412(6845):449-52
references.pmid: 11473321
references.citation: PLoS Biol. 2003 Nov;1(2):E41
references.pmid: 14624243