Figure 1.

Overexpressed Tcb3-GFP Localizes to the Dot-like Domains of Perinuclear ER, and Its Localization Depends on C2 Domains and Tcb1p/Tcb2p

(A) Diagram of domain organization of Tcb3 protein. Amino acid length is indicated. TMD, transmembrane domain; SMP, synaptotagmin-like mitochondrial lipid-binding protein; C2, calcium-dependent lipid-binding domain.

(B) Endogenously expressed Tcb3-GFP localizes to cortical ER (cER) in wild-type cells. Cells expressing Tcb3p tagged with GFP at the C terminus under the control of TCB3 endogenous promoter were grown at 25°C and imaged by differential interference contrast (DIC) and fluorescence microscopy. Scale bar, 5 μm.

(C–G) The C2 domains are required for Tcb3p localization to the dot-like domains of perinuclear ER (nER). tcb3Δ cells overexpressing Tcb3-GFP (C) or Tcb3(ΔSMP.C2)-GFP (D) under TDH3 promoter were grown at 25°C and imaged by DIC and fluorescence microscopy. An arrow in the image in C indicates dot-like structures. Scale bar, 5 μm. Bottom panels are intensity plots along the white line in C and D. nER/cER intensity ratios for Tcb3-GFP, Tcb3(ΔSMP)-GFP, Tcb3(ΔC2)-GFP, and Tcb3(ΔSMP-C2)-GFP (E), percentages of cells with GFP dots (F) were quantified, and distribution of Tcb3-GFP dot was shown (G). The data represent the mean ± standard deviation (SD) of three independent experiments, each based on more than 100 cells. ∗∗p < 0.01 and ∗∗∗p < 0.001 by Student's t-test. n.s., not significant.

(H and I) Tcb1p and Tcb2p are required for formation of Tcb3-GFP-positive dot structures. nER/cER intensity ratios for tcb3Δ and tcb1Δ tcb2Δ tcb3Δ cells expressing Tcb3-GFP under TDH3 promoter (H) and quantification of percentages of cells with GFP dots (I). The data represent mean ± SD of three independent experiments, each based on more than 100 cells. ∗p < 0.05 by Student's t-test. n.s., not significant.