Figure 8.
SpSpo13 specifically facilitates nucleotide exchange by SpYpt2. (A) SpSpo13 stimulates GDP release by SpYpt2. 400 nM SpYpt2-mant-GDP was used per reaction. At the time point indicated by the arrow, GST-ScSec21-160 or GST-SpSpo13 was added to the reaction mixture at the indicated concentrations. (B) SpSpo13 cannot stimulate GDP release by SpYpt1 or SpYpt3. We used 400 nM SpYpt1-mant-GDP or 400 nM SpYpt3 per reaction. At the time point indicated by the arrow, 400 nM GST-SpSpo13 or 6 mM EDTA was added to the reaction mixture. (C) SpSpo13 genetically interacts with SpYpt2 in vivo. Strain HJ75-4 (sec2Δ; pCEN-URA3-ScSEC2) was cotransformed with a CEN-TRP1-plasmid carrying Spspo13+ and 2μ-LEU2-plasmids carrying indicated YPT genes. The transformed strains were grown in nonselective media overnight, and 10-fold serial dilutions of the overnight culture were spotted onto a synthetic plate lacking tryptophan and leucine or onto a plate containing 5-fluoroorotic acid (5-FOA). The plates were photographed after 2-d incubation at 30°C.