Fig. 6. AtKSS protein interactions. (A) Prey proteins isolated from the yeast two-hybrid screen. (B,C) Yeast two-hybrid interactions. Growth of strains on minimal SD media lacking tryptophan (TRP) and leucine (LEU) (left). ß-galactosidase assay of a replica of the left panels (center). Growth of yeast strains on minimal SD media lacking TRP, LEU, adenine (ADE) and histidine (HIS) (right). (B) Fusion proteins expressed from the DNA-binding (BD) and activation (AD) domains: 1 (BD: AtKSS; AD: 1.38); 2 (BD: AtKSS; AD: 1.25); 3 (BD: AtKSS; AD: 1.25B2); 4 (BD: AtKSS; AD: KSN1); 5 (BD: AtKSS; AD: 1.86); 6 and 8 (positive controls, Clontech); 7 (negative control, Clontech). (C) Protein-protein interaction assays using empty BD vectors. Yeast strains 1 to 5 had the AD as in B but carried the empty pGBDT7 BD vector. Yeast strains 6 to 8 were as in B. (D) Co-immunoprecipitation of AtKSS and prey proteins (left panel) using in vitro methionine ³⁵S-labelled translated proteins (right panel). Proteins were incubated in the presence of either anti-HA or anti-c-Myc antibodies. Protein complexes were pulled down using protein-G-coupled Dynabeads. Polypeptides for in vitro translation were: AtKSS (p60): PGABKT7-AtKSS; KTN p80.1 (p80): clone pGADT7-1.25B2; KSN1: clone pGADT7-1.52; LAMIN C: pGBKT7-Lam (Clontech). (E) Sequence alignment of the C-terminal regions of putative katanin p80 proteins. Amino acids residues conserved in at least five sequences are in black boxes, similar residues are in gray. At-1 to At-4, Arabidopsis AAB71474, CAC08339, AAD49999 and BAB09559. Os-1 and Os-2, rice BAB63574 and BAB91860. Hs-1, human XP_048046. Mm, mouse BAB26884. Xl-1, Xenopus laevis AAC25113. Sp-1, Strongylocentrotus purpuratus AAC09329. The consensus (cons.) is presented beneath the alignment. The underlined sequence represents the polypeptide encoded by clone 1.25B2.

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