Histone deacetylase 8 interacts with the GTPase SmRho1 in Schistosoma mansoni
Fig 5
The interaction between SmRho1.1 and SmHDAC8 is dependent on the SmRho1.1 C-terminus.
(A) Y2H mating experiments showed that SmHDAC8 interacts specifically with SmRho1.1 protein. AH109 yeasts expressing only Gal4AD (pGADT7) or Gal4AD-fused SmRho1.1 or SmRho1.2 were mated with Y187 yeasts expressing only Gal4DBD (pGBKT7) or Gal4DBDfused SmHDAC8. Diploids were allowed to grow on a minimal SD -Leu/-Trp medium (left panel) and diploids expressing interacting proteins were then selected on SD -Leu/-Trp/-His/Ade medium (right panel). Only yeasts expressing SmHDAC8 and SmRho1.1 grew on the selective medium. The results presented are from one experiment representative of three carried out. (B) SmHDAC8 binds only SmRho1.1 but not SmRho1.2. Co-IP and WB analysis of SmHDAC8 and SmRho1 isoforms expressed in X. laevis oocytes showed an interaction only between SmHDAC8 (Myc-tagged) and SmRho1.1 (HA-tagged). cRNAs encoding HA-tagged SmRho1.1 or SmRho1.2 were co-injected in X. laevis oocytes with cRNA encoding Myc-tagged SmHDAC8. Oocytes were incubated in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated by anti-HA or anti-Myc antibodies and analyzed by WB to detect SmHDAC8- Myc (50 kDa) and SmRho1-HA isoforms (22 kDa) with anti-Myc or anti-HA antibodies. Experiments were repeated three times on oocytes from three different females. (C) Schematic structure of SmRho1.1 and SmRho1.2 mutants. Using site-directed mutagenesis, the glutamic acid Glu33 of SmRho1.1 was substituted by a lysine (SmRho1.1 E33K) and the lysine Lys33 of SmRho1.2 by a glutamic acid (SmRho1.2 K33E). SmRho1. 1–143 aa and SmRho1. 1–88 aa proteins are portions of SmRho1.1. produced by site-directed mutagenesis. (D) Co-IP and WB experiments performed in X. laevis oocytes revealed that SmRho1. 1–143 aa and SmRho1. 1–88 aa mutants (HA-tagged) are not able to bind SmHDAC8 (Myc -tagged). cRNAs encoding HA-tagged SmRho1 isoforms, SmRho1.1 mutant or SmRho1.2 mutants were co-injected in X. laevis oocytes with cRNA encoding Myc-tagged SmHDAC8. Oocytes were incubated in ND96 medium and lysed. Proteins from soluble extracts were immunoprecipitated (IP) by anti-HA or anti-Myc antibodies and analyzed by WB to detect SmHDAC8 (50 kDa), SmRho1 isoforms (22 kDa) or SmRho1 mutants (22kDa) with anti-Myc or anti-HA antibodies. SmHDAC8 co-immunoprecipitated only with SmRho1.1 E33K.