Figure 4.
Proteins Encoded by Parologous Copies of the Pollen-Expressed AhSLF Genes Do Not Interact with S-RNases in Yeast.
(A) Constructs made for the full-length, N-terminal, and C-terminal regions of AhSLF-S1E, AhSLF-S2C, and AhSLF-S4D in pGBTK7 plasmid and expressed as fusions to GAL4 DNA binding domains, which are represented by an X. AD::S-RNases were as described in Figure 2.
(B) Growth of yeast strains transformed with various combinations of BD and AD plasmids on -Leu/-Ade media. S1E, AhSLF-S1E; S2C, AhSLF-S2C; S4D, AhSLF-S4D; S2, AhSLF-S2. Plus signs indicate the normal growth of the transformed yeast strains.
(C) Growth of AH109 yeast strains transformed with various combinations of BD and AD plasmids on -Trp/-His/-Leu/-Ade media. No growth occurred except BD::AhSLF-S2 and AD::S-RNases. Minus signs indicate no growth of the transformed yeast strains.
(D) Expression of the S-RNase fusion proteins were assayed by immunoblot analysis with the anti-S-RNase antibody in transformed yeast cells. The S2-RNase (lanes 1, 4, and 7), S4-RNase (lanes 2, 5, and 8), and S5-RNase (lanes 3, 6, and 9) fusion proteins were expressed properly in the transformed yeast cells. Lane 10 represents style proteins of S2S5.
(E) Expression of the AhSLF fusion proteins were assayed by immunoblot analysis with the anti-Myc antibody in transformed yeast cells. Lanes 1 to 10 represent AhSLF-S1E-N, -S1E-C, -S1E-F, -S2C-N, -S2C-C, -S2C-F, -S4D-N, -S4D-C, -S4D-F, and -S2-C fusion proteins expressed in the transformed yeast cells, respectively. Suffixes -C, -N, and -F denote the C-terminal, N-terminal, and full-length products, respectively.