Yagoub Daniel, Hart-Smith Gene, Moecking Jonas, Erce Melissa A, Wilkins Marc R
Systems Biology Laboratory, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
Proteomics. 2015 Sep;15(18):3209-18. doi: 10.1002/pmic.201500075. Epub 2015 Aug 10.
The Hmt1 methyltransferase is the predominant arginine methyltransferase in Saccharomyces cerevisiae. There are 18 substrate proteins described for this methyltransferase, however native sites of methylation have only been identified on two of these proteins. Here we used peptide immunoaffinity enrichment, followed by LC-ETD-MS/MS, to discover 21 native sites of arginine methylation on five putative Hmt1 substrate proteins, namely Gar1p (H/ACA ribonucleoprotein complex subunit 1), Nop1p (rRNA 2'-O-methyltransferase fibrillarin), Npl3p (nucleolar protein 3), Nsr1p (nuclear localization sequence-binding protein), and Rps2p (40S ribosomal protein S2). The sites, many of which were found to be mono- or di-methylated, were predominantly found in RGG (Arg-Gly-Gly) motifs. Heavy methyl-SILAC validated the majority of these peptides. The above proteins, and relevant sites of methylation, were subsequently validated by in vitro methylation with recombinant Hmt1. This brings the total of Hmt1 substrate proteins for which native methylation sites have been identified to five.
Hmt1甲基转移酶是酿酒酵母中主要的精氨酸甲基转移酶。该甲基转移酶有18种底物蛋白，但仅在其中两种蛋白上鉴定出了天然甲基化位点。在此，我们采用肽免疫亲和富集，随后进行液相色谱-电子转移解离串联质谱（LC-ETD-MS/MS），以发现5种假定的Hmt1底物蛋白上的21个精氨酸甲基化天然位点，这5种蛋白分别是Gar1p（H/ACA核糖核蛋白复合体亚基1）、Nop1p（rRNA 2'-O-甲基转移酶纤维蛋白原）、Npl3p（核仁蛋白3）、Nsr1p（核定位序列结合蛋白）和Rps2p（40S核糖体蛋白S2）。这些位点大多被发现为单甲基化或双甲基化，主要存在于RGG（精氨酸-甘氨酸-甘氨酸）基序中。重甲基化稳定同位素标记（Heavy methyl-SILAC）验证了这些肽段中的大多数。上述蛋白及相关甲基化位点随后通过用重组Hmt1进行体外甲基化得以验证。这使得已鉴定出天然甲基化位点的Hmt1底物蛋白总数达到了5种。
