An expanded family of TPC genes in animals. (A) Maximum likelihood tree constructed using the conserved regions of TPC sequences from the representative organisms listed in Table S1. Shading highlights TPC isoforms in plants, protists, and animals, with the latter subdivided into three distinct groupings. Bootstrap values >50 are shown at the branches. (B) End point RT-PCR analysis showing expression of transcripts for TPC isoforms in the indicated cell type/embryo (prism stage). The expected sizes of the amplicons were 152 (SpuTPC1), 93 (SpuTPC2), 107 (SpuTPC3), 473 (RnoTPC1), 575 (RnoTPC2), 250 (HsaTPC1), and 250 bp (HsaTPC2). Black lines indicate that intervening lanes have been spliced out. (C) Quantitative RT-PCR of TPC isoforms. Data were normalized to the expression level of the indicated housekeeping gene. Error bars indicate SEM.

Overexpression of TPC1 enhances NAADP-mediated calcium signals. (A–C) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with either buffer (A) or 10 nM NAADP (B and C). Cells were from mock-transfected cultures (black) or cultures expressing TPC1 mRFP (green). In C, cells were pretreated with 1 µM bafilomycin A1 (Baf) for 60 min or 10 µM ryanodine (Ry) for 15 min as indicated. Data are expressed as mean fluorescence ratios from 4–11 cells. (D) Pooled data quantifying the magnitude of the ratio changes under the various experimental conditions. Error bars indicate SEM.

Mutation of a single residue in the putative pore region inactivates TPC1. (A) Multiple sequence alignment of the two putative pore regions (P1 and P2) of several animal TPCs. Asterisks highlight residues conserved in both pores of all isoforms from different species. Predicted helical region is outlined by the cartoon. (B and C) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with either 10 nM (B) or 10 µM (C) NAADP. Cells were from mock-transfected cultures or cultures expressing TPC1 mutated in the putative pore region (TPC1 L273P). (D) Pooled data quantifying the magnitude of cytosolic changes under the various experimental conditions are shown. Error bars indicate SEM.

TPCs are intracellular proteins targeted to the endolysosomal system. (A and B) Confocal images of X. laevis oocytes coexpressing a plasma membrane marker (human reduced folate carrier [RFC]–GFP; green) and either TPC1-mRFP (A) or TPC2-mRFP (B). Images were taken at the level of the plasma membrane (i), cortical ER (ii), and subcortical ER (iii). Bars, 30 µm. (C and D) Higher magnification images of TPC1-mRFP (C) and TPC2-GFP expression (D). Insets show a digitally zoomed region. Plots of fluorescence intensity from regions indicated by the arrows are shown below. Bars, 10 µm. (E and F) Confocal images of SKBR3 cells coexpressing TPC1 (E) or TPC2 (F) tagged with either mRFP or GFP (top row) and organelle markers (middle row) for lysosomes (Lyso)/late endosomes (LAMP1-mRFP; left column), early and late endosomes (endo-GFP; middle column), or the ER (DsRed2-KDEL; right column). Overlays of the images are shown in the bottom rows. Intensity plots of green and red fluorescence across the regions delimited by the arrows in the overlay images are shown below. Bar, 5 µm.

Knockdown of TPC1 reduces NAADP-mediated calcium signals. (A) Cytosolic calcium responses of individual fura-2–loaded SKBR3 cells microinjected with 10 µM NAADP. Cells were from mock-transfected cultures (black) or cultures expressing either a control (Ctrl) shRNA (blue) or an shRNA-targeting TPC1 (red). (B) Pooled data quantifying the magnitude of the ratio changes under the various experimental conditions. Error bars indicate SEM.