Amino acids affect Rim15 activity.

<p>(A) Wild type DBY746, was transformed with a plasmid coding for the Rim15-GFP fusion protein, after two days of growth the exhausted medium was replaced (upper panel) with fresh synthetic medium lacking all the amino acids (dextrose) or with the complete medium (SDC), pictures were taken after the indicated time. In the lower panel the fresh medium contained also compounds necessary to compensate the auxothrophies (the amino acids L, H, W and the nucleotide Uracil) plus the indicated amino acid (pictures were taken after two hours of incubation). (B) Wild type (Wt) and <i>sch9Δ</i> mutants (<i>sch9Δ</i>) were transformed with a plasmid coding for the Rim15-GFP fusion protein. After two days of growth wild type cells were transferred onto fresh medium while sch9Δ cells were left in the exhausted medium. GFP fluorescence or DAPI staining were observed after 2 additional hours in the aforementioned medium. (C) Wild type DBY746 strain, transformed with Rim15-GFP and Pab1-RFP expressing plasmids was grown to the post-diauxic phase (two days of growth) and then transferred onto fresh complete SDC medium, fluorescence was observed after two hours of incubation. (D) Rim15 mRNA level fluctuation in response to nutrients addition. mRNA obtained from stationary phase DBY746 cell culture (T = 0) or from the same culture but collected after 1, 2 or 4 hours after the switch to fresh SDC was subjected to Real Time PCR with Rim15 specific oligonucleotides. The amount of mRNA used for each PCR was normalized using the actin (ACT1) transcript levels as an internal control. The expression level of Rim15 before the medium switch (T = 0), which was the highest, was used as a reference point (100% of expression). The expression at the others time points was calculated using the formula [expr level(T = 1, 2, 4)/expr level (T = 0)] * 100.