Nature. 2010 Jul 28. [Epub ahead of print]
NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein.
Schaner Tooley CE, Petkowski JJ, Muratore-Schroeder TL, Balsbaugh JL, Shabanowitz J, Sabat M, Minor W, Hunt DF, Macara IG.
Department of Microbiology, Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
The post-translational methylation of alpha-amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins L16 and L33 (refs 1, 2), but almost nothing is known about the function or enzymology of this modification. Several other bacterial and eukaryotic proteins have since been shown to be alpha-N-methylated. However, the Ran guanine nucleotide-exchange factor, RCC1, is the only protein for which any biological function of alpha-N-methylation has been identified. Methylation-defective mutants of RCC1 have reduced affinity for DNA and cause mitotic defects, but further characterization of this modification has been hindered by ignorance of the responsible methyltransferase. All fungal and animal N-terminally methylated proteins contain a unique N-terminal motif, Met-(Ala/Pro/Ser)-Pro-Lys, indicating that they may be targets of the same, unknown enzyme. The initiating Met is cleaved, and the exposed alpha-amino group is mono-, di- or trimethylated. Here we report the discovery of the first alpha-N-methyltransferase, which we named N-terminal RCC1 methyltransferase (NRMT). Substrate docking and mutational analysis of RCC1 defined the NRMT recognition sequence and enabled the identification of numerous new methylation targets, including SET (also known as TAF-I or PHAPII) and the retinoblastoma protein, RB. Knockdown of NRMT recapitulates the multi-spindle phenotype seen with methylation-defective RCC1 mutants, demonstrating the importance of alpha-N-methylation for normal bipolar spindle formation and chromosome segregation.
I really enjoy reading this paper - classic biochemistry experiments and elegant MS analysis, from Macara group in UVA.
Love it!
The following I think is the key to this success!
- Good antibodies: anti-me2-SPK (Ser-Pro-Lys), anti-me3-SPK, anti-me2-PPK (Rabbit anti-me2-PPK antibody was produced against 2me-P-P-K-R-I-A-K-R-R-S-C synthesized by American Peptide.)
- In vitro methylation assays. Substrate proteins were expressed with C-terminal His6 tags, and assayed in 50mM Tris, 50mM potassium acetate, pH8.0, with 100 mM SAM as the methyl donor. All reactions were incubated 1 h at 30 C and analysed formethylation byimmunoblot. For ELISAassays, substrates and enzyme were mixed with 0.55 mCi 3H-S-adenosyl-L-methionine (SAM). Reactions were incubated as above then transferred to Protein A-coated ELISA plates pretreated with anti-me2-SPK. After 1 h, the reaction was removed and wells were treated with 1% SDS for 30 min. This solution was then quantified for incorporated 3Hmethyl by scintillation counting.
- Screening mutant NRMT substrates. Efficient removal of the initiating Met is dependent on the identity of the second residue. To avoid this problem, we created fusions from residues 2–10 of RCC1 with His6–tag plus Factor X cleavage site at the N terminus, and C-terminal GFP. Cleavage with Factor X exposes the second residue of the RCC1 Nterminus. Substrate proteins containing all possible amino acids at residue 2 were expressed in Escherichia coli and purified on Ni-NTA beads, then cleaved using Factor X.
- LC-MS/MS identification of novel NRMTsubstrates. On-bead endoproteinase Glu-C and Asp-N digestions were used to identify the immunoprecipitated proteins. The retained N termini were then eluted from the beads and analysed for methylation (Supplementary Fig. 5).
NICE~~
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