Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 6, Pages 1439-1451Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.011680
Keywords
phosphorylation; protein phosphorylation; cell signaling; post-translational modification (PTM); molecular cell biology; inorganic polyphosphate; lysine modifications; lysine polyphosphorylation (K-PPn); polyphosphatases; Top1
Categories
Funding
- European Union's Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie Grant Agreement PHEMDD. [752903]
- German Research Foundation (DFG) under Germany's Excellence Strategy [CIBSS-EXC-2189, 390939984]
- Medical Research Council (MRC) [MC_UU_00012/4]
- Marie Curie Actions (MSCA) [752903] Funding Source: Marie Curie Actions (MSCA)
- MRC [MC_UU_12018/4, MC_UU_00012/4] Funding Source: UKRI
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A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1?ppn2?, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain.
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