FIG 9.

Characterization of the Asp1 interaction partner Met10. (A) Yeast two-hybrid analysis of the interaction between Asp1 and Met10 (SPCC584.01c). S. cerevisiae strain AH109 was cotransformed with a plasmid expressing asp1⁺ fused to the GAL4 binding domain (pGBKT7) and a plasmid expressing an met10 variant (amino acids 544 to 1006) fused to the GAL4 activation domain (pGADT7). Cells were spotted on plasmid-selective SD medium with or without histidine and incubated for 5 days at 30°C. (B) Growth of wild-type and met10Δ strains on the indicated medium: full medium (YE5S), minimal medium (MM), MM plus 330 μM cysteine (MM+Cys), MM plus 140 μM methionine (MM+Met), or MM plus cysteine and methionine (MM+Cys+Met). (C) Serial dilution patch tests (10⁴ to 10¹ cells) of a wild-type strain transformed with a vector (control) or plasmid expressing asp1⁺ or met10⁺ from the nmt1⁺ promoter. Transformants were grown at 25°C for 8 days. (D) Serial dilution patch tests (10⁴ to 10¹ cells) of transformed asp1Δ cells with a vector (control) or plasmid expressing asp1⁺ or met10⁺ via nmt1⁺. Incubation was performed at 25°C for 11 days. (E) Far-Western analysis. A Coomassie-stained gel of 1 μg of the indicated purified proteins on the far left. Blots, from left to right, show the following: protein-protein interaction of GST-Met10 (blotted protein; 138 kDa, arrow) and Asp1^365–920-His (probe protein) using His antibody for detection of GST-Met10; interaction of Asp1^365–920-His (blotted protein) and GST (probe protein) with a GST antibody (control); protein-protein interaction of Asp1^365–920-His (blotted protein; 65 kDa, arrow) and GST-Met10 (probe protein) using GST antibody for detection of Asp1^365–920-His. One microgram of protein was loaded on the gel in all cases. Concentration of probe proteins, 10 μg/ml.