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. 2022 Oct 20;17(10):e0274908. doi: 10.1371/journal.pone.0274908

Fig 5. Viability of Spf1p and M1 mutated Spf1p.

Fig 5

A) Alignment of M1 region of Spf1p and similar P5A ATPase sequences. Conserved residues F202, F204 and F207 indicated by star and predicted α-helical structure shown in blue. B) Calculated ΔG (kcal/mol) for folding of M1 and M2 in Spf1p and M1 mutated spf1p. Introduction of histidines, but not alanines, increase the calculated energetic requirement for proper folding into the α-helical structure. C) Complementation in spf1 deletant cells using the expression constructs used in this study. The catalytic dead mutations D487A and E310A and the empty vector was used as a control. Complementation was tested on 10 mM caffeine plates as described in [12]. D) Specific activity of purified Spf1p mutants in mixed YPL/OG micelles. All experiments are performed as biological replicates and technical triplicates (n = 6) and is reported as the average value of these with error bars indicating the standard error. E) Expression analysis of the constructs used in this study, SDS-PAGE show expression of a ~130 kDa band corresponding to Spf1 and M1 mutated Spf1p. A western blot against the FLAG-tag using Anti-FLAG AB is shown for reference. F) SDS-PAGE gels of a typical purification showing relatively pure and homogenous preparations comparable to those presented in [12]. Fractions containing Spf1p were pooled for recovery of purified Spf1p.