Open Access Research article

VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase

Yorann Baron, Patrick G Pedrioli, Kshitiz Tyagi, Clare Johnson, Nicola T Wood, Daniel Fountaine, Melanie Wightman and Gabriela Alexandru*

Author Affiliations

Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU), College of Life Sciences, University of Dundee, Dow St, Dundee DD1 5EH, UK

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BMC Biology 2014, 12:39  doi:10.1186/1741-7007-12-39

Published: 29 May 2014

Additional files

Additional file 1: Table S1:

Mass spectrometry analysis of Flag-FAF1 immunoprecipitates from human U2OS cells. Anti-Flag immunoprecipitates from untransfected cells were used as a negative control. Protein coverage and the share of spectrum IDs are indicated for each protein identified in the immunoprecipitates.

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Additional file 2: Figure S1:

Ubiquitinated forms of VAPB are not detectable in Flag-VAPB immunoprecipitates. U2OS cells expressing Flag-VAPB from a tetracycline-inducible promoter were induced by addition of 100 ng/ml tetracycline. Flag-VAPB was immunoprecipitated after treatment with 10 μM MG132 for 1 or 2 hr, or 5 μM for 6 hr or from untreated cells (0 hr). Two exposures of Flag-VAPB immunoblots are shown. No ubiquitinated forms of Flag-VAPB can be detected with or without proteasome inhibition, not even after a very long exposure.

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Additional file 3: Table S2:

Canonical FFAT proteins do not accumulate in endogenous VAPB immunoprecipitates upon proteasome inhibition.

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Additional file 4: Table S3:

SILAC mass spectrometry analysis of endogenous VAPB immunoprecipitates from human U2OS cells. Light- or heavy-labeled cells were treated with MG132 for 2 or 6 hr, as indicated. Equal amounts of light- and heavy-labeled extracts were mixed and endogenous VAPB was immunoprecipitated using specific antibodies. The light/heavy SILAC ratios (L/H) determined by mass spectrometry are indicated, as well as the protein coverage. L + MG indicates that the light-labeled samples were treated with MG132. Proteins whose interaction with VAPB is stimulated by proteasome inhibition accumulate in these samples, resulting in L/H ratios higher than 1. H + MG indicates that the heavy-labeled samples were treated with MG132. Protein accumulation in the heavy-labeled samples results in L/H ratios lower than 1. The L/H for proteins that are not affected by proteasome inhibition will be close to 1.

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Additional file 5: Table S4:

Mass spectrometry analysis of Flag-FAF1 immunoprecipitates from human U2OS cells treated with MG132 for 0, 2 or 6 hr as indicated. Anti-Flag immunoprecipitates from untransfected cells were used as a negative control. Protein coverage and the share of spectrum IDs are indicated for each protein identified in the immunoprecipitates.

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Additional file 6: Table S5:

Ubiquitinated targets of VAPB and FAF1 identified by mass spectrometry upon enrichment of ubiquitinated peptides. A mixture of light-labeled Flag-VAPB and heavy-labeled Flag-FAF1 immunoprecipitates was analyzed by mass spectrometry after ubiquitinated peptide enrichment using antibodies specific to lysine-ϵ-GlyGly. The peptides containing lysine residues with an additional MW due to the GlyGly modification – 250.15 (heavy label) or 242.14 (light label) – are indicated for each protein.

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Additional file 7: Table S6:

Mass spectrometry analysis of Flag-VAPA/B immunoprecipitates from human U2OS cells. Anti-Flag immunoprecipitates from untransfected cells were used as a negative control. Protein coverage and the share of spectrum IDs are indicated for each protein identified in the immunoprecipitates.

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Additional file 8: Table S7:

Mass spectrometry analysis of endogenous VAPB immunoprecipitates from human HeLa cells. For the negative control sample, cell extracts were incubated with uncoupled Protein A-beads and the proteins retained on these beads were analyzed by mass spectrometry. Protein coverage and the share of spectrum IDs are indicated for each protein identified.

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Additional file 9: Table S8:

Mass spectrometry analysis of endogenous VAPB immunoprecipitates from mouse brain. For the negative control sample, brain extracts were incubated with uncoupled Protein A-beads and the proteins retained on these beads were analyzed by mass spectrometry. Protein coverage and the share of spectrum IDs are indicated for each protein identified.

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Additional file 10: Figure S2:

STX1A and B are not FFAT-like proteins. (A) Endogenous VAPB interacts with STX1A in mouse brain. (B) Alignment of the sequences that resemble FFAT motifs in human STX1A and B. (C) Flag-STX1A or B mutated for the two phenylalanine residues (F33A-F34A and F32A-F33A, respectively) in the putative FFAT motifs interact with VAPB similar to their WT counterparts. IP, immunoprecipitate; WT, wild type.

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