FIGURE 2.
TRF1 binding and telomere localization of a large panel of PinX1 mutants. A, TRF1 binding and telomere localization of single point mutants or quadruple Ala substitution mutants (K292A, K294A, K295A, and R297A) in different PinX1 fragments were summarized, respectively. B and C, examples of TRF1 binding of selected PinX1 mutants are shown. Selected single point mutations or quadruple Ala mutation (K292A, K294A, K295A, and R297A) were introduced into full-length PinX1 or various PinX1 fragments, and mutant proteins were expressed in bacteria and purified as GST fusion proteins followed by GST pulldown with in vitro synthesized 35S-TRF1. The amino acid residues of PinX1 truncation mutants are from 254 to 328 (TID), from 291 to 328 (C37), and from 291 to 310 (C20). D, examples of subcellular localization of TID or TID4A with TRF1 are shown. The localization of these mutant proteins were determined by co-transfecting HT1080 cells overnight with vectors expressing RFP-tagged TRF1 and GFP-tagged wild-type full-length PinX1 or its TID with or without mutations followed by subjecting them to fluorescence microscopy (D). Yellow arrows point to colocalization of TRF1 and TID at telomeres.