Figure 8.

Leucine 140 is found in a conserved hydrophobic region. (A) Multiple sequence alignment of Rpn3 from a variety of fungal species. Leucine 140 (S. cerevisiae) is indicated by an asterisk. The alignment was performed with MUSCLE (Edgar 2004). In order, the proteins are as follows: Aspergillus nidulans, XP_660371.1; Saccharomyces cerevisiae, NP_010938.1; Candida glabrata, XP_447606.1; Kluyveromyces lactis, XP_453544.1; Zygosaccharomyces rouxii, XP_002496514.1; Lachancea thermotolerans, XP_002554976.1; Ashbya gossypii, NP_983112.1; Lodderomyces elongisporus, XP_001526739.1; Pichia guilliermondii, XP_001487379.1; and Scheffersomyces stipitis, XP_001383755.2. (B) Multiple sequence alignment of Rpn3 from a variety of eukaryotes. The proteins are as follows: Schizosaccharomyces pombe, NP_595282.2; Drosophila melanogaster, NP_477300; Xenopus laevis, NP_001085955.1; Mus musculus, NP_033465.1; and Homo sapiens, NP_002800.2. (C) Expression of Rpn3-L140P. Extracts were prepared from BY4741 expressing myc⁹-Rpn3 (lanes 1-3) or myc⁹-Rpn3-L140P from a centromeric plasmid; 50, 25, and 10 μg of protein was separated by sodium dodecyl sulfate (SDS)-PAGE and Western-blotted with anti-Myc antibody (upper panel). The lower section of the gel was stained with Coomassie brilliant blue (lower panel). (D) Phenotype of rpn3-L140P in isolation. Yeast strains BY4741 (RPN3) and CY6398 (rpn3-L140P) were grown to stationary phase and serial dilutions were spotted onto YPD plates at 30° and 37° and onto a minimal plate containing 1.0 mg/l canavanine. (E) rpn3-L140P does not suppress tra1-F3744A. Yeast strains CY4353 (TRA1 RPN3), CY4350 (tra1-F3744A RPN3), and CY6418 (tra1-F3744A rpn3-L140P) were grown to stationary phase and serial dilutions were spotted onto YPD plates at 30° and 37° or a YPD plate containing 6% ethanol at 30°.