Abstract
Arginine (arg) methylation is a widespread posttranslational modification of proteins that impacts numerous cellular processes such as chromatin remodeling, RNA processing, DNA repair, and cell signaling. Known arg methylproteins arise mostly from yeast and mammals, and are almost exclusively nuclear and cytoplasmic. Trypanosoma brucei is an early branching eukaryote whose genome encodes five putative protein arg methyltransferases, and thus likely contains a plethora of arg methylproteins. Additionally, trypanosomes and related organisms possess a unique mitochondrion that undergoes dramatic developmental regulation and uses novel RNA editing and mitochondrial DNA replication mechanisms. Here, we performed a global mass spectrometric analysis of the T. brucei mitochondrion to identify new arg methylproteins in this medically relevant parasite. Enabling factors of this work are use of a combination digestion with two orthogonal enzymes, an efficient offline two dimensional chromatography separation, and high-resolution mass spectrometry analysis with two complementary activations. This approach led to the comprehensive, sensitive and confident identification and localization of methylarg at a proteome level. We identified 167 arg methylproteins with wide-ranging functions including metabolism, transport, chaperoning, RNA processing, translation, and DNA replication. Our data suggest that arg methylproteins in trypanosome mitochondria possess both trypanosome-specific and evolutionarily conserved modifications, depending on the protein targeted. This study is the first comprehensive analysis of mitochondrial arg methylation in any organism, and represents a significant advance in our knowledge of the range of arg methylproteins and their sites of modification. Moreover, these studies establish T. brucei as a model organism for the study of posttranslational modifications.
Highlights
Arginine1 methylation is a widespread post-translational modification with roles in numerous cellular functions such as chromatin remodeling, RNA processing, DNA repair, and cell signaling [1]
Mitochondria Contain a Distinct Set of Arg Methylproteins—We previously showed that arg methylation of a trypanosome mitochondrial RNA binding protein, RBP16, affects its macromolecular interactions and ability to stabilize specific RNAs [18, 23], suggesting that arg methylation could be more widespread in this organelle
All four antibodies reveal a pattern of methylproteins in mitochondria that is distinct from that detected by the same antibody in whole cell extract
Summary
Cells—Procyclic form (PF) T. brucei strain EATRO 164 Istar was cultured as described [16]. Separation was performed on a Thermo Scientific BioBasic SCX column (4.6 ϫ 250 mm, 5-m particle size) at a flow rate of 1 ml/min, with an injection volume of 600 l, which contains tryptic or GluC peptides derived from 1 mg of mitochondrial proteins. To achieve a comprehensive separation of the complex peptide mixture, a nano-LC/nanospray setup, which features low void volume and high chromatographic reproducibility [22], was employed. A stringent set of criteria were employed to filter the data, including 15 ppm precursor mass tolerance and high Xcorr and delta-CN cut-off values (XcorrϾ1.8 for 1ϩ charge, Ͼ2.1 for 2ϩ charge, Ͼ3 for 3ϩ charge and Ͼ4 for 4ϩ charge, and delta-CNϾ0.1) that resulted in a peptide FDR of 0.3%. For each methylated peptide identified by ETD, the corresponding CID spectrum was manually inspected to determine the symmetry of the methylation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
