Fig 9.

Introduction of H134 is sufficient to allow PTTG2 to bind separase. (A) PTTG2^R134H gains ESP1 binding capability. A schematic diagram of PTTG2 point mutants used in this study is shown. R¹³⁴ and G¹⁵³ of PTTG2 (black) were mutated to the corresponding sequences in PTTG1 (gray) either individually or together. HeLa cells were transiently transfected with ESP1- and FLAG-tagged wild-type or mutant PTTG2, as indicated. Binding to ESP1 was detected by immunoprecipitation with FLAG antibodies, followed by elution and immunoblotting. (B) PTTG1 and PTTG2 are differently located. HeLa cells stably expressing histone H2B-GFP were cotransfected with plasmids expressing the indicated FLAG-tagged constructs and histone H2B-GFP. Cells were processed for immunostaining with antibodies against FLAG. The histone H2B signals indicate the nucleus. (C) PTTG2^R134H interferes with normal mitosis. HeLa cells were transfected with plasmids expressing the indicated constructs. Transfected cells were differentiated by virtue of a cotransfected histone H2B-GFP plasmid. The cells were harvested at the indicated time points and analyzed by flow cytometry. (D) Nondegradable PTTG2^R134H prevents sister chromatid separation. HeLa cells expressing histone H2B-GFP and APC/C biosensor were transfected with plasmids expressing a DΔ version of PTTG1, PTTG2, or PTTG2^R134H. A histone H2B-mRFP-expressing construct was used as a cotransfection marker. Individual cells were then tracked for 24-h time-lapse microscopy. Each horizontal bar represents one cell (n = 50). Light gray, interphase; black, mitosis (from DNA condensation to anaphase or cell death or DNA decondensation without anaphase); dark gray, interphase after DNA decondensation without anaphase; truncated bars, cell death. (E) Still images from a representative PTTG1^DΔ-expressing mitotic cell. Channels of histone H2B-GFP and mRFP-APC/C biosensor are shown. The full video can be found as Video S6 in the supplemental material.