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. 2011 Jun 22;156(4):1783–1796. doi: 10.1104/pp.111.177816

Figure 1.

Figure 1.

TPK1-GFP is located at the tonoplast in transgenic Arabidopsis. A, Predicted topology for AtTPK1, with four TMDs and two P domains. The N- and C-terminal domains contain a 14.3.3 protein binding site and two putative EF hands, respectively. B, Proteins extracted from leaves of wild type (WT) or three T2 plants expressing TPK1-GFP from transformation event number 9 (lanes 1–3) were analyzed by SDS-PAGE and protein blot using anti-GFP antiserum. The positions of TPK1-GFP (black arrowhead) and free GFP (white arrowhead) are indicated at right. Numbers at left indicate the positions of molecular mass markers, in kD. C, Leaves (top and center sections) or roots (bottom sections) were analyzed by epifluorescence microscopy. In each lane, bright-field (left) GFP fluorescence (center) and merge of the two (right) are shown. Arrows indicate plasma membranes. D, Subcellular compartments were fractionated by ultracentrifugation of leaf homogenate on isopycnic Suc gradient. Proteins in each fraction were analyzed by SDS-PAGE and protein blot with antibodies against endoplasmin (Endop., ER marker), PIP2 (plasma membrane marker), γ-TIP (tonoplast marker), or GFP (to detect TPK1-GFP). Top of the gradient is at left; numbers on top indicate fraction density (g/mL); P indicates the precipitate recovered from the bottom of the tube.