title: VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase.
abstract: BACKGROUND
  FAF1 is a ubiquitin-binding adaptor for the p97 ATPase and belongs to the UBA-UBX family of p97 cofactors. p97 converts the energy derived from ATP hydrolysis into conformational changes of the p97 hexamer, which allows the dissociation of its targets from cellular structures or from larger protein complexes to facilitate their ubiquitin-dependent degradation. VAPB and the related protein VAPA form homo- and heterodimers that are anchored in the endoplasmic reticulum membrane and can interact with protein partners carrying a FFAT motif. Mutations in either VAPB or p97 can cause amyotrophic lateral sclerosis, a neurodegenerative disorder that affects upper and lower motor neurons.
  
  
  RESULTS
  We show that FAF1 contains a non-canonical FFAT motif that allows it to interact directly with the MSP domain of VAPB and, thereby, to mediate VAPB interaction with p97. This finding establishes a link between two proteins that can cause amyotrophic lateral sclerosis when mutated, VAPB/ALS8 and p97/ALS14. Subsequently, we identified a similar FFAT-like motif in the ASNA1 subunit of the transmembrane-domain recognition complex (TRC), which in turn mediates ASNA1 interaction with the MSP domain of VAPB. Proteasome inhibition leads to the accumulation of ubiquitinated species in VAPB immunoprecipitates and this correlates with an increase in FAF1 and p97 binding. We found that VAPB interaction with ubiquitinated proteins is strongly reduced in cells treated with FAF1 siRNA. Our efforts to determine the identity of the ubiquitinated targets common to VAPB and FAF1 led to the identification of RPN2, a subunit of an oligosaccharyl-transferase located at the endoplasmic reticulum, which may be regulated by ubiquitin-mediated degradation.
  
  
  CONCLUSIONS
  The FFAT-like motifs we identified in FAF1 and ASNA1 demonstrate that sequences containing a single phenylalanine residue with the consensus (D/E)(D/E)FEDAx(D/E) are also proficient to mediate interaction with VAPB. Our findings indicate that the repertoire of VAPB interactors is more diverse than previously anticipated and link VAPB to the function of ATPase complexes such as p97/FAF1 and ASNA1/TRC.
authors.forename: Yorann
authors.initials: Y
authors.lastname: Baron
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Pedrioli
authors.forename: Kshitiz
authors.initials: K
authors.lastname: Tyagi
authors.forename: Clare
authors.initials: C
authors.lastname: Johnson
authors.forename: Nicola T
authors.initials: NT
authors.lastname: Wood
authors.forename: Daniel
authors.initials: D
authors.lastname: Fountaine
authors.forename: Melanie
authors.initials: M
authors.lastname: Wightman
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU), College of Life Sciences, University of Dundee, Dow St, Dundee DD1 5EH, UK. g.alexandru@dundee.ac.uk.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Alexandru
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins; D053501:Arsenite Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D006602:Hexosyltransferases; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C490780:FAF1 protein, human; C489667:GET3 protein, human; D009687:Nuclear Proteins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; C114051:VAPA protein, human; C496748:VAPB protein, human; D033921:Vesicular Transport Proteins; D006602:Hexosyltransferases; C517590:RPN2 protein, human; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C404942:p97 ATPase; D053501:Arsenite Transporting ATPases; C580955:RAB3GAP1 protein, human; D020692:rab3 GTP-Binding Proteins