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. 2021 Dec 14;9(12):2584.
doi: 10.3390/microorganisms9122584.

YPK9 and WHI2 Negatively Interact during Oxidative Stress

Florenal Joseph  1 , Darach Miller  2   3 , Oleg V Evgrafov  4 , William J Chirico  1   4
Affiliations

YPK9 and WHI2 Negatively Interact during Oxidative Stress

Florenal Joseph et al. Microorganisms. .

Abstract

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson's disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741COM). In contrast, deleting YPK9 from BY4741COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741COM and BY4741COMypk9∆ contain a novel premature stop codon near the 3' end of WHI2 (WHI2G1324T), whereas the collection's YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2pE442*) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2G1324T strain's genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

Keywords: ATP13A2; Parkinson’s disease; Saccharomyces cerevisiae; hydrogen peroxide; yeast.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
H2O2 sensitivity of the YKO collection and laboratory YPK9 deletion strains. The growth curves of BY4741COM (squares), the YKO collection’s ypk9∆ in BY4741 (circles), and our ypk9∆ in BY4741COM (triangles) were compared in the presence (solid symbols) or absence (open symbols) of 50 µM H2O2. Differences in growth (see text) were based on OD600 at mid-log point. Error bars represent the SEM, N = 3.
Figure 2
Figure 2
Alignment of Whi2p variants. The reference strain (S288C), BY4742, and YKO collection’s ypk9∆ contain the sequence for full-length Whi2p (486 amino acids). In contrast, BY4741COM’s WHI2 sequence has a premature stop codon yielding a truncated Whi2p of 441 amino acids residues. Clustal Omega was used to align sequences [29].
Figure 3
Figure 3
Sequence confirmation of engineered WHI2 variants. A hygromycin cassette was fused to WHI2 or its variant containing a stop codon at position 1324 (WHI2G1324T) and used to replace the endogenous version of WHI2 in BY4741COM, BY4742, and YKO collection’s ypk9∆ in BY4741YKO. DNA sequencing was used to confirm the replacement. The asterisk represents the stop codon. Numbers with a hashtag represent the laboratory strain identification number.
Figure 4
Figure 4
Effect H2O2 on the growth of WHI2 and YPK9 variants in different background strains. (A) WHI2 variants in the YKO collection’s ypk9∆ in BY4741YKO. (B) WHI2 variants in BY4741COM. (C) WHI2 and YPK9 variants in BY4742. Hygromycin (Hyg) was used to select for gene replacement. The OD shown is the maximum recorded during 25 h of growth. Results were analyzed using one-way ANOVA and Tukey’s post hoc test. Error bars represent the SEM, n = 3. Asterisks represent p-values: * 0.03332, ** 0.0021, **** < 0.0001; ns, not significant.
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References

    1. Gitler A.D., Chesi A., Geddie M.L., Strathearn K.E., Hamamichi S., Hill K.J., Caldwell K.A., Caldwell G.A., Cooper A.A., Rochet J.-C., et al. Alpha-Synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity. Nat. Genet. 2009;41:308–315. doi: 10.1038/ng.300. - DOI - PMC - PubMed
    1. Schmidt K., Wolfe D.M., Stiller B., Pearce D.A. Cd2+, Mn2+, Ni2+ and Se2+ toxicity to Saccharomyces cerevisiae lacking YPK9p the orthologue of human ATP13A2. Biochem. Biophys. Res. Commun. 2009;383:198–202. doi: 10.1016/j.bbrc.2009.03.151. - DOI - PMC - PubMed
    1. van Veen S., Martin S., Van den Haute C., Benoy V., Lyons J., Vanhoutte R., Kahler J.P., Decuypere J.-P., Gelders G., Lambie E., et al. ATP13A2 deficiency disrupts lysosomal polyamine export. Nature. 2020;578:419–424. doi: 10.1038/s41586-020-1968-7. - DOI - PubMed
    1. Ramirez A., Heimbach A., Gründemann J., Stiller B., Hampshire D., Cid L.P., Goebel I., Mubaidin A.F., Wriekat A.-L., Roeper J., et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat. Genet. 2006;38:1184–1191. doi: 10.1038/ng1884. - DOI - PubMed
    1. Tabor C.W., Tabor H. Polyamines in microorganisms. Microbiol. Rev. 1985;49:81–99. doi: 10.1128/mr.49.1.81-99.1985. - DOI - PMC - PubMed

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