Figure 5: An additional N77K exchange suppresses the phenotypes of drg1-21.

(a) Domain representation of Drg1 with the location of the Drg1-21 exchanges (T100I and L108P) and the additional suppressor exchange (N77K, magenta). (b) Drg1 knockout strains expressing the drg1-21^sup allele (N77K/T100I/L108P) or the drg1-N77K allele (N77K) as control were spotted on SD-leu as well as on SD + 5-FOA to confirm plasmid shuffling and were incubated at 25 °C and 37 °C. (c) The N77K exchange partially restores the regulation of ATPase activity of the Drg1-21 variant. Drg1 wildtype as well as mutant variants were purified and the in vitro ATPase activity was measured using the Malachite green phosphate assay. The ATPase activity was determined in the absence (basal activity) and in the presence (stimulated activity) of the HIS₆-tagged C-terminal fragment of the substrate protein Rlp24 (amino acids 147-199). The ATPase activity was measured for proteins purified in the presence or absence (w/o) of 1 mM ADP. All values are presented as specific ATPase activity (μmol ATP h⁻¹ mg⁻¹ Drg1). Error bars represent standard deviations of means calculated from at least two biological replicates each measured in triplicate. P-values were calculated by an unpaired t-test (n.s.: not significant; p > 0.05)