Deciphering the role of N‐terminal methylation in modulating yeast protein function including the multitasking stress response protein, Hsp31

Abstract

The first cytosolic N‐terminal methyltransferase identified in yeast, Tae1, recognizes mainly a protein N‐terminal motif sequence of M‐X‐P‐K. This conserved sequence motif is found in multiple proteins including ribosomal proteins, histones and small heat shock proteins across species. The human homolog of Tae1, N‐terminal methyltransferase 1 (NTMT1/NRMT1) has important functions in cell division by regulating RCC1 and centromere proteins such as CENP‐A and is implicated as a cancer target. Although these studies have been informative, the biological function of alpha N‐terminal methylation in yeast and human remains largely unknown. Our research is directed at unravelling the role of N‐terminal methylation in regulating yeast protein function and is currently focused on Hsp31, a multifunctional chaperone and enzyme involved in oxidative stress response. We are also investigating N‐terminal methylation on a proteome‐wide basis.Immuno‐detection and tandem mass spectrometry (MS/MS) assay with overexpressed Hsp31‐ MORF or physiologically expressed Hsp31‐GFP purified from WT and Tae1 deficiency strains demonstrated that Hsp31‐MORF is methylated at the first alanine (initial methionine is efficiently removed by a native methionine aminopeptidase) after the protein is synthesized. We also observed that the methylation types are exclusively mono‐ or di‐ methylation, with no tri‐methylated species observed. This is the first demonstration that the Hsp31 chaperone protein is N‐terminally methylated. Interestingly, methylation of Hsp31 was decreased but not completely eliminated in Tae1 deficient strains. This is the first evidence of such an overlapping methyltransferase activity which suggests the presence of another enzyme with N‐terminal methyltransferase activity. It unclear how the biological function of Hsp31 is affected by methylation and the difference between mono‐/di‐ methylation and tri‐methylation. We also verified N‐terminal methylation on other potential Tae1 substrates by MS/MS, including Hsp33 and a xylose and arabinose reductase. We conclude that N‐terminal methylation occurs with diverse substrates and that yeast is an excellent model to probe the function of this post‐translational modification. Further investigation is ongoing to determine if and how N‐terminal methylation modulates Hsp31 functions using site‐directed mutagenesis and biological activity assays.Support or Funding InformationFunding: Purdue University MCMP Research Enhancement grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.