Figure 4.
SpSpo13 facilitates nucleotide exchange on ScSec4. (A) SpSpo13 stimulates GTP binding. ScSec4 was preloaded with GDP. At t = 300 s (indicated by arrow), ScSec4 alone, ScSec4 mixed with GST, ScSec4 mixed with GST-ScSec21-160, or ScSec4 mixed with GST-SpSpo13 was added to buffer containing the fluorescent GTP analogue mant-GppNHp. We used 400 nM ScSec4-GDP per reaction. Binding of the analogue to ScSec4 was monitored by following the fluorescence signal created by a FRET interaction between ScSec4 and the mant-GppNHp. The gray line indicates the basal level of fluorescence signal from the fluorophore under these conditions. (B) SpSpo13 stimulates GDP release. 6XHis-ScSec4 preloaded with the fluorescent GDP analogue mant-GDP was incubated in buffer containing GppNHp. We used 400 nM ScSec4-mant-GDP per reaction. At the time indicated by the arrow, buffer, GST-ScSec21-160, or GST-SpSpo13 was added to the reaction mixture. Release of GDP was monitored by a loss of fluorescent signal from the ScSec4-mant-GDP FRET interaction. The gray line indicates the basal level of fluorescence signal from the fluorophore under these conditions.