Discovery of Arginine Methylation, Phosphorylation, and Their Co-occurrence in Condensate-Associated Proteins in Saccharomyces cerevisiae

The
formation of condensates in membraneless organelles is thought
to be driven by protein phase separation. Arginine methylation and
serine/threonine phosphorylation are important in the phase separation
process; however, these post-translational modifications are often
present in intrinsically disordered regions that are difficult to
analyze with standard proteomic techniques. To understand their presence
and co-occurrence in condensate-associated proteins, here, we use
a multiprotease and multi-tandem mass spectrometry (MS/MS) fragmentation
approach, coupled with heavy methyl stable isotope labeling of amino
acids in cell culture (SILAC) and phospho- or methyl-peptide enrichment.
For Saccharomyces cerevisiae, we report
a 50% increase in the known arginine methylproteome, involving 15
proteins that are all condensate-associated. Importantly, some of
these proteins have arginine methylation on all predicted sitesproviding
evidence that this modification can be pervasive. We explored whether
arginine-methylated, condensate-associated proteins are also phosphorylated
and found 12 such proteins to carry phosphorylated serine or threonine.
In Npl3, Ded1, and Sbp1, single peptides were found to carry both
modifications, indicating a co-occurrence in close proximity and on
the same protein molecule. These co-modifications occur in regions
of disorder, whereas arginine methylation is typically on regions
of disorder that are also basic. For phosphorylation, its association
with charged regions of condensate-associated proteins was less consistent,
although some regions with multisite phosphorylation sites were strongly
acidic. We conclude that arginine-methylated proteins associated with
condensates are typically also modified with protein phosphorylation.