FIGURE 5.

Identification of a region in the C terminus of tTG that is crucial for its ability to assume the closed conformation. Linear representations showing the relative lengths of Myc-tagged forms of tTG WT, tTG-Short, and a tTG 1–672. The numbers shown indicate amino acids. B, cell lysates collected from NIH3T3 fibroblasts ectopically expressing the vector alone or one of the indicated Myc-tagged tTG proteins for ∼24 h were immunoblotted (IB) with Myc and actin antibodies. C, NIH3T3 cells ectopically expressing the vector alone or one of the indicated Myc-tagged tTG proteins were cultured in medium containing 10% CS for ∼36 h and fixed. Immunofluorescence was performed on the cells using a Myc antibody to detect the transfectants, and the cells were also stained with DAPI to label nuclei. Cells undergoing apoptosis were identified by nuclear condensation/blebbing. The experiment was performed three separate times, and the percent apoptosis for each condition was determined by calculating the ratio of apoptotic to non-apoptotic cells. The data shown represent the mean ± S.E. D, the C-terminal 15 amino acids of tTG (residues 673–687) are highlighted in purple in the x-ray crystal structure of tTG bound to GDP (top panel). Two pairs of hydrogen bonds formed between residues Asn-681 and Asp-434 and between residues Lys-677 and Trp-254 are shown in magenta with participating residues shown in sticks (bottom panels). The distances of each interaction are shown.