Fig. 4. Mouse PINS interacts with the asymmetric localization domain of Drosophila Insc through its TPR 3-7. (A) Yeast two-hybrid assays show that interaction between PINS and full-length Insc (FL-Insc) is mediated through the TPR repeats of Pins (T1-7 represents a construct containing all seven TPRs, T3-T7 represents a construct containing TPR 3 to 7, etc.). The interaction activities between Insc and various parts of PINS are semi-quantitated based on the time taken for colonies to turn blue in X-gal filter lift assay: ++, 30-90 minutes; +, >120 minutes; -, no significant staining. (B) PINS can interact specifically with the asymmetric localization domain of Insc (Insc-5, aa 288-497) as well as full-length Insc and Insc-2 (aa 258-578), which also contains the asymmetric localization domain, but not N-terminal Insc (N-Insc, aa 1-330) lacking this domain of Insc. In vitro translated [³⁵S]-labeled full-length PINS was incubated with sepharose 4B beads coupled to GST and various GST-Insc fusion proteins. PINS is able to bind to all GST-Insc fusion proteins (Fl-Insc, aa 1-859; Insc-5, aa 288-497; Insc-2, aa 258-578) containing the asymmetric localization domain of Insc (aa 288-497) but not to GST alone nor to N-terminal Insc (N-Insc, aa1-330), which lacks the asymmetric localization domain. (C,D) To further characterize this interaction, various [³⁵S]-labeled portions of PINS (N-PINS: aa 1-369; C-PINS: aa 366-672; T3-7: aa 129-369; T3-6: aa 129-315; T4-7: aa 182-369 and T3-5: aa 129-275) were incubated with sepharose 4b beads coupled to the full-length Insc GST fusion protein or coupled to GST alone. Like Drosophila Pins, N-PINS containing the TPR interacts with Insc, whereas C-PINS does not interact (C). The region TPR3-7 of PINS can be pulled down by Insc but TPR3-5 and TPR4-7 can not (D). Although a trace mount of TPR3-6 is pulled down by Insc, the region TPR 3-6 does not interact with Insc in yeast two-hybrid assay, suggesting that TPR7 is necessary for the interaction.