Identification of known and novel components of mRNA transport complexes by mass spectrometry. (A) Venn diagram showing the number of proteins classed as enriched on the ILS, HLE, and TLS localization signals by MS/MS using our selection criteria (>4 normalized spectral counts [nSCs] present on WT signal and >80% of the total normalized spectral counts for a WT and paired mutant signal present on the WT signal; nSCs are spectral counts normalized to correct for differences in the total number of spectra between individual MS runs; see Materials and methods and Table S3 legend for details). Proteins were only classed as enriched on the ILS if they fulfilled the selection criteria in the two independent experiments. Proteins classed as enriched on the TLS in A are from the comparison of the WT element to the mutant ΔCA, which has a stronger inhibitory effect than the U6C mutant on recruitment of known components of the localization machinery (B). (B) nSCs observed for the 11 proteins enriched on all three localization signals tested. The nSCs for the two biological replicates of ILS vs. ILS^as experiments are shown separately for comparison, as are the data for the TLS^U6C mutant. Note that differences in nSCs between different proteins are not a good reflection of abundance, as these values are heavily influenced by the protein’s molecular mass and how well individual peptides are detected by MS. (C) Immunoblot showing the enrichment of Dic, Dlic, p50^Dmn, CLIP-190, and Lis1 on active localization signals. The Lis1-interacting protein NudE is not detectable on any signal.