To abolish ERM binding without affecting the α-actinin binding site, only the first three key residues R, T and Y of the ERM binding motif were mutated to alanines.
We constructed a kinase-dead JNK1- DN mutant K55R, in which lysine-55, which is critical for phosphotransfer in the ATP-binding motif, is mutated to arginine and generated stable transfectants in ZE cells.
Anti-GFP immunoprecipitations were performed on extracts from cells expressing either GFP-OCRLWT (wild type OCRL) or GFP-OCRLX3 (an OCRL mutant in which both clathrin boxes as well as the AP-2-binding motif are mutated [ Mao et al., 2009]).
The C-ter domain has on its own a marked affinity for heparin that decreases dramatically when HS-binding motifs are mutated.
This nuclear localization is more pronounced when the RNA-binding motifs were mutated 15.
When the clathrin-binding FDPL motif was mutated in mouse Tom1L1, the mutant failed to rescue the endocytosis of EGFR in the knockdown cells (lane 4), whereas another mutant altering DLQP region (which is not involved in CHC interaction, Figure 7D, lane 5) was still able to rescue the endocytosis.
A peptide nanowire of 33 amino acids, comprised of a leucine zipper motif, was mutated to bind divalent metals, conferring conductivity into the peptide.
Therefore, we generated a GFP-CXXCDnmt1 construct where the KFGG motif was mutated to AAGG (GFP-CXXCDnmt1KF/AA, Figure S4 in File S1) to test the requirement of the KFGG motif for binding by the CXXC domain of Dnmt1.
The effect of ouabain on the T55 reporter varied weakly when a single "AUUUA" motif was mutated, but was almost completely suppressed when all "AUUUA" motifs were mutated (Fig 6B, right panel).
Finally, in vitro pulldowns with recombinant taperin and PP1α show a robust interaction, again abolished when the putative PP1 binding KISF motif is mutated (Fig. 1D), establishing this as the primary PP1 dock site on taperin.
