substrate proteins were expressed with c - terminal his6 tags , and assayed in 50 mm tris , 50 mm k acetate , ph 8.0 , with 100 m sam as the methyl donor .
all reactions were incubated 1 h at 30c and analyzed for methylation by immunoblot . for elisa assays ,
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 .
efficient removal of the initiating met is dependent on the identity of the second residue . to avoid this problem , we created fusions from residues 210 of rcc1 with his6-tag plus factor x cleavage site at the n - terminus , and c - terminal gfp .
xpk - egfp substrate proteins were expressed in e.coli and purified on ni - nta beads , then cleaved using factor x. 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 analyzed for methylation ( supplementary fig .
substrate proteins were expressed with c - terminal his6 tags , and assayed in 50 mm tris , 50 mm k acetate , ph 8.0 , with 100 m sam as the methyl donor .
all reactions were incubated 1 h at 30c and analyzed for methylation by immunoblot . for elisa assays ,
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 .
efficient removal of the initiating met is dependent on the identity of the second residue . to avoid this problem , we created fusions from residues 210 of rcc1 with his6-tag plus factor x cleavage site at the n - terminus , and c - terminal gfp .
xpk - egfp substrate proteins were expressed in e.coli and purified on ni - nta beads , then cleaved using factor x.
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 analyzed for methylation ( supplementary fig .
 the post - translational methylation of -amino groups was first discovered over 30 years ago on the bacterial ribosomal proteins l16 and l3312 , 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 -n - methylated310 . however , the ran guanine nucleotide - exchange factor , rcc1 , is the only protein for which any biological function of -n - methylation has been identified3 , 11 . 
 methylation - defective mutants of rcc1 have reduced affinity for dna and cause mitotic defects3 , 11 , 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 they may be targets of the same , unknown enzyme3,12 . 
 the initiating met is cleaved , and the exposed -amino group is mono- , di- , or trimethylated . 
 here we report the discovery of the first -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 / taf - i / phapii and the retinoblastoma protein , rb . 
 knockdown of nrmt recapitulates the multi - spindle phenotype seen with methylation - defective rcc1 mutants3 , demonstrating the importance of alpha - n - methylation for normal bipolar spindle formation and chromosome segregation . 
METHODS SUMMARY
None
Screening mutant NRMT substrates
LS-MS/MS identification of novel NRMT substrates
Supplementary Material