The green fluorescent pHluorin is quenched at lower pH values ( 5.6), whereas the DsRed fluorescence remains bright. were monitored after sequential additions of propionic acid (30 s) followed Vatiquinone by NH4Cl (1 min 30 s). The assay was followed for approximately 5 min, and the movie was generated using 6 fps at slow speed. Download Movie S2, MOV file, 9.8 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S1. Detection of HAL and PCR diagnostics of knock-in mutant cell lines. (A) Western blot analysis to detect HAL and its mutated version in protein lysates from yeast mutants (parental cell line (WT) and HA-tagged cell lines (C1 and C2) resolved by NuPAGE gel electrophoresis. Detection was performed using mouse anti-HAL (1:5,000) or anti-HA (1:2,000) as detailed in Materials and Methods. Arrows indicate monomeric sizes of HAL and HAL-3HA (60 kDa). (D) Effect of polyP130 on activity of the 6His-HAL-C13 mutant. Bars represent changes in fluorescence obtained in the linear region of histidine conversion under saturating Mouse monoclonal to COX4I1 conditions after preincubation (2 min) with polyP, as indicated in Fig.?5B but using a larger excitation slit width (10 nm). Download FIG?S1, TIF file, 2.9 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 Vatiquinone International license. FIG?S2. PolyP analysis in yeast mutants expressing HAL. (A) Total polyP extracted from GFP-HAL and GFP yeast strains and analyzed by PAGE and toluidine blue staining. (B) Equal amounts (50 g) of total polyP were extracted from WT-background polyP mutants and resolved by PAGE followed by toluidine blue staining. PolyP20, polyP100 (2 mM), and inositol hexakisphosphate (IP6) were used as standard references to compare with samples as indicated above the gels. OG, orange G. Download FIG?S2, TIF file, 2.5 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. TABLE?S1. Oligonucleotides used for genome editing and cloning procedures for HAL-pH, yeast assays, and mutagenesis. Download Table?S1, PDF file, 0.10 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 Vatiquinone International license. TABLE?S2. Yeast strains generated in this study. Download Table?S2, PDF file, 0.1 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. VIDEO?S3. Glutamate/histidine-driven alkalinization after acidic pulse. Live imaging was recorded in parasites seeded onto polylysine-coated slides, and changes in fluorescence (DsRed and pHluorin green) were monitored after sequential additions of propionic acid (30 s) followed by l-glutamate (1 min 30 s) and l-histidine (3 min). The assay was followed for approximately 5 min, and the movie was generated using 6 fps at medium speed. Download Movie S3, MOV file, 15.2 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Vatiquinone Attribution 4.0 International license. VIDEO?S4. Parasite burst after alkaline stress. Live imaging was recorded in parasites seeded onto polylysine coated slides, and changes in fluorescence (DsRed and pHluorin green) were monitored after sequential additions of NH4Cl (30 s) followed by l-histidine (1 min 30 s). The assay was followed for approximately 5 min, and the movie was generated using 6 fps at medium speed. Download Movie S4, MOV file, 14.3 MB. Copyright ? 2021 Mantilla et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. ABSTRACT and conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell.