A Screen for Candidate Targets of Lysine Polyphosphorylation Uncovers a Conserved Network Implicated in Ribosome Biogenesis

Abstract

Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.

Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast

“…While the sequence requirements for polyP addition are unclear, lysine residues appear to be the modified site (7,9,10). Consistent with this, mutation of lysine residues within predicted PASK motifs to arginine prevents polyphosphorylation of Nsr1, Top1, and Rpa34 (7,9). Polyphosphorylation causes dramatic electrophoretic shifts when protein extracts are run on denaturing Bis-Tris NuPAGE gels, but this same shift does not occur with traditional SDS-PAGE gels (7,9).…”

“…Polyphosphorylation is non-enzymatic and occurs in polyacidic serine and lysine rich (PASK) motifs (7,8). While the sequence requirements for polyP addition are unclear, lysine residues appear to be the modified site (7,9,10). Consistent with this, mutation of lysine residues within predicted PASK motifs to arginine prevents polyphosphorylation of Nsr1, Top1, and Rpa34 (7,9).…”

“…Polyphosphorylation causes dramatic electrophoretic shifts when protein extracts are run on denaturing Bis-Tris NuPAGE gels, but this same shift does not occur with traditional SDS-PAGE gels (7,9). The underlying reasons for this difference are not completely clear, although it could be related to the use of TEMED in the polymerization of SDS-PAGE gels (9).…”

“…Recently, we reported the identification of an additional 15 polyphosphorylation targets in yeast, including a conserved network implicated in ribosome biogenesis (9). Many of the PASK motifs in these proteins are contained within intrinsically disordered regions that appear to be under evolutionary selection (11).…”

“…Many of the PASK motifs in these proteins are contained within intrinsically disordered regions that appear to be under evolutionary selection (11). Based on enrichment in ribosome-related functions, we uncovered a novel function for VTC4, encoding the catalytic subunit of the VTC complex (12), in polysome assembly (9). We also demonstrated that 6 human proteins, including homologs of our novel yeast targets, could be polyphosphorylated following the ectopic expression of E. coli PPK, the bacterial polyP polymerase, in HEK293T cells (9).…”