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General Information
Name
Rab_Effectors_1
Species
D. melanogaster
Result type
FlyBase ID
FBlc0000723
Project
Data Provider
Title
Unfiltered Rab protein interactions derived from 18 GST-Rab pull downs and mass spectrometry.
Status
Current
Accessions
    Biosample Source
    Overview
    Strain
    Stage
    Sex
    Tissue isolated
    Other tissues studied
    Cell component
    Key genes
    Methods
    Sample preparation

    For each Rab column, 5E8 S2 cells were harvested by centrifugation for lysis.

    Biosamples analyzed by this result (1)
    Biosample
    Type
    Title
    D. melanogaster, S2 cell line, source of protein extract for GST pull downs.
    Data Analyzed
    Assay methods
    Key genes
    Protocol

    Each of the 23 D. melanogaster Rabs that have a mammalian ortholog were expressed as recombinant GST fusions (in E. coli) bearing the Q>L mutation that is known to stabilize the GTP-bound form (constitutively active). GST-Rab fusions were coupled to glutathione sepharose and incubated with clarified S2 cell lysates (10-20mg/ml protein extract) that were prepared using CHAPS detergent, in the presence of non-hydrolysable GTP analogue GppNHp. Columns were washed and bound proteins were eluted using high salt and GDP. Proteins were concentrated by chloroform/methanol protein precipitation, separated by SDS-PAGE, and proteins >45 KDa were excised for subsequent identification.

    Mode of Assay

    Purified proteins were identified by liquid chromatography-mass spectrometry. Peptide searches were against FlyBase Dmel 5.9.

    Raw Data Analyzed (1)
    Assay / Reagent collection
    Type
    Title
    Pull down of proteins in S2 cell extract using GST-Rab protein as bait, identification by mass spectrometry.
    Processed Data Analyzed (0)
    Result
    Type
    Title
    Analysis
    Methods
    Reference Genome
    Reference Annotation
    Data analysis

    Peptide searches were against FlyBase Dmel 5.9, and protein identifications were accepted if they could be established at >95.0% probablility and contained at least two identified peptides. The predicted false discovery rate (FDR) was less than 1%. Spectral counts were used to estimate protein abundance. An "S-score" was calculated for each interaction, which gives greater weight to interactions seen with fewer baits.

    Comments

    The use of detergent during S2 cell lysis solubilized more proteins, which allowed for the recovery of more known Rab interacting proteins, but also increased the number of non-specifing binding proteins isolated across the parallel affinity purifications. Using detergent-free cell lysis in the related Rab_Effectors_2 dataset resulted in a cleaner dataset, but some known Rab-binding proteins were lost or reduced.

    No specific interactions were recovered for Rab3, Rab8, Rab10, Rab21, Rab23, Rab27, Rab32 or Rab40.

    Associated Data
    Size
    Associated interactions
    146 Interactions
    Files
    Additional Information
    Synonyms and Secondary IDs (3)
    Reported As
    Symbol Synonym
    Rab interactome detergent data set
    Rab_Effectors_1
    Name Synonyms
    Unfiltered Rab protein interactions derived from 18 GST-Rab pull downs and mass spectrometry.
    Secondary FlyBase IDs
      References (2)