E suspect that thisTesting Co-localization with TRPMLAs another approach to validate ITI-007 supplier Candidate TRPML1 interactors, we assayed co-localization of the identified proteins with GFP-TRPML1 in RAW264.7 cells. GFP-TRPML1 predominantly Tubastatin A chemical information localizes to late endosome and lysosomes of these murine macrophages at steady state, similar to GFP-TRPML1’s localization in other cell types [19]. While we only analyzed the cells that expressed minimal levels of fusion proteins that were still detectable by microscopy, we cannot rule out that some colocalization with GFP-TRPML1 may be a consequence of the overexpression of the fusion proteins. Some TagRFP(S158T)-fused candidate proteins were not detectable by microscopy either because the steady state levels of these proteins were too low in the transfected cells and/or because the TagRFP(S158T) epitope affects the folding and hence stability of the fusion proteins. For these proteins, we used the V5-fused forms and performed immunofluorescence analysis on the cells to assay co-localization with GFP-TRPML1; the V5 epitope (GKPIPNPLLGLDST) is relatively small and is hence less likely to interfere with the folding of the fusion protein.Figure 4. Split-Ubiquitin Yeast Two-Hybrid Tests of Candidate Interactors. The same number of cells of yeast 
strains carrying indicated constructs were spotted on SD eu rp (LT) plates that select for plasmids or SD eu rp de ?his +1 mM 3-AT (LTAH) plates that assay for interaction. Fur4-NubG, Ost1-NubG, and NubG are negative controls; Fur4-NubI and Ost1-NubI are positive controls. doi:10.1371/journal.pone.0056780.gProteins That Interact with TRPMLFigure 5. Co-Localization Tests of Candidate Interactors. A, Plasmids expressing TagRFP(S158T) or V5 fusions to candidate interactors were transfected into RAW264.7 macrophages that stably express GFP-TRPML1. Confocal microscopy was done on fixed cells. Cells transfected with V5-X proteins were immunostained to localize the V5 fusion proteins. B, Quantitation of percent of TagRFP(S15T)/V5-X discrete structures that also have GFP-TRPML1. Bars represent standard deviations. doi:10.1371/journal.pone.0056780.gOf the candidate interactors identified by the Immunoprecipitation/Mass Spectrometry screen, TRPML1 showed significant co-localization with PEA-15, STOML1, Rac2, Cdc42, RhoG (but not Rac1), and NP9 (Fig. 5; Table 1). Of the candidate interactors identified by the SU-YTH screen, TRPML1 showed a low level of co-localization with YIF1 1662274 and BAE30441 (Fig. 5; Table 1).DiscussionWe describe two large-scale screens for TRPML1 interactors, the first based on Immunoprecipitation/Mass Spectrometry and the second using SU-YTH assays. Each of these screens identified a list of potential TRPML1 interactors with minimal overlap. The only protein identified by both screens was isoform 3 of glyceraldehyde 3-phosphate dehydrogenase, but the screens also identified homologous proteins for the alpha subunit of a sodium channel protein and for cadherin-like proteins. To determine the validity of the Immunoprecipitation/Mass Spectrometry and the SU-YTH screens, we carried out anunbiased survey of some potential interactors identified by each screen. Of seven proteins tested from the Immunoprecipitation/ Mass Spectrometry list, four proteins, Rac2, Cdc42, NP9, and STOML1, are strong candidate interactors of TRPML1, showing association with TRPML1 using both Immunoprecipitation/ Western, either at endogenous or elevated levels, and SU-YTH assays (Table 1). O.E suspect that thisTesting Co-localization with TRPMLAs another approach to validate candidate TRPML1 interactors, we assayed co-localization of the identified proteins with GFP-TRPML1 in RAW264.7 cells. GFP-TRPML1 predominantly localizes to late endosome and lysosomes of these murine macrophages at steady state, similar to GFP-TRPML1’s localization in other cell types [19]. While we only analyzed the cells that expressed minimal levels of fusion proteins that were still detectable by microscopy, we cannot rule out that some colocalization with GFP-TRPML1 may be a consequence of the overexpression of the fusion proteins. Some TagRFP(S158T)-fused candidate proteins were not detectable by microscopy either because the 
steady state levels of these proteins were too low in the transfected cells and/or because the TagRFP(S158T) epitope affects the folding and hence stability of the fusion proteins. For these proteins, we used the V5-fused forms and performed immunofluorescence analysis on the cells to assay co-localization with GFP-TRPML1; the V5 epitope (GKPIPNPLLGLDST) is relatively small and is hence less likely to interfere with the folding of the fusion protein.Figure 4. Split-Ubiquitin Yeast Two-Hybrid Tests of Candidate Interactors. The same number of cells of yeast strains carrying indicated constructs were spotted on SD eu rp (LT) plates that select for plasmids or SD eu rp de ?his +1 mM 3-AT (LTAH) plates that assay for interaction. Fur4-NubG, Ost1-NubG, and NubG are negative controls; Fur4-NubI and Ost1-NubI are positive controls. doi:10.1371/journal.pone.0056780.gProteins That Interact with TRPMLFigure 5. Co-Localization Tests of Candidate Interactors. A, Plasmids expressing TagRFP(S158T) or V5 fusions to candidate interactors were transfected into RAW264.7 macrophages that stably express GFP-TRPML1. Confocal microscopy was done on fixed cells. Cells transfected with V5-X proteins were immunostained to localize the V5 fusion proteins. B, Quantitation of percent of TagRFP(S15T)/V5-X discrete structures that also have GFP-TRPML1. Bars represent standard deviations. doi:10.1371/journal.pone.0056780.gOf the candidate interactors identified by the Immunoprecipitation/Mass Spectrometry screen, TRPML1 showed significant co-localization with PEA-15, STOML1, Rac2, Cdc42, RhoG (but not Rac1), and NP9 (Fig. 5; Table 1). Of the candidate interactors identified by the SU-YTH screen, TRPML1 showed a low level of co-localization with YIF1 1662274 and BAE30441 (Fig. 5; Table 1).DiscussionWe describe two large-scale screens for TRPML1 interactors, the first based on Immunoprecipitation/Mass Spectrometry and the second using SU-YTH assays. Each of these screens identified a list of potential TRPML1 interactors with minimal overlap. The only protein identified by both screens was isoform 3 of glyceraldehyde 3-phosphate dehydrogenase, but the screens also identified homologous proteins for the alpha subunit of a sodium channel protein and for cadherin-like proteins. To determine the validity of the Immunoprecipitation/Mass Spectrometry and the SU-YTH screens, we carried out anunbiased survey of some potential interactors identified by each screen. Of seven proteins tested from the Immunoprecipitation/ Mass Spectrometry list, four proteins, Rac2, Cdc42, NP9, and STOML1, are strong candidate interactors of TRPML1, showing association with TRPML1 using both Immunoprecipitation/ Western, either at endogenous or elevated levels, and SU-YTH assays (Table 1). O.
