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. Author manuscript; available in PMC: 2022 Jul 7.
Published in final edited form as: Nat Cell Biol. 2021 Feb 11;23(3):243–256. doi: 10.1038/s41556-021-00631-y

Extended Data Fig. 2 |. Phosphorylation by Tor2/TORC1 stabilizes Pir1.

Extended Data Fig. 2 |

a, Immunopurified fractions from cells expressing untagged or GFP-tagged Pir1 (left) and FLAG-tagged Tor2 (right) were subjected to mass spectrometry. The total PSM and coverage for the identified proteins is shown. b, Multiple sequence alignment of S. pombe proteins Pir1 and Red1 against human ZFC3H1 was performed using Macaw sequence alignment workbench. Pir1 aligns with ZFC3H1 in their shared serine/proline rich region. Thick rectangles indicate segments of best alignment and dotted lines indicate gaps. Alignments for two conserved regions are shown in detail. The serines that are phosphorylated in Pir1 are labelled with orange triangles. S285 is in a conserved motif which is underlined in red. Mass spectrometry results are from a single experiment. c, Phosphorylation of Xpress-tagged recombinant WT Pir1 and mutant Pir1 with 12 serines (S215, S233, S237, S248, S265, S285, S299, S303, S325, S334, S434, S503) substituted with alanine by the FLAG–Tor2 complex was detected using anti-thiophosphate-ester antibody. d, Western blot analysis of GFP–Pir1 in the indicated strains at 30 °C. 12 SD, 12 serines replaced by aspartic acid. e, Western blot analysis of Red1 in the indicated strains at 30 °C. SD, serine285 replaced by aspartic acid. For c-e, two independent experiments were performed with similar results. Source data are provided.