N‐Terminal methylation of proteasome subunit Rpt1 in yeast

The 26 S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20 S proteasome) as well as regulatory particles, which contain six ATP ase ( R pt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20 S proteasome. In this study, we used MS to analyze the N ‐terminal modifications of the yeast R pt1 subunit, which contains the N ‐terminal recognition sequence for N ‐methyltransferase. Our results revealed that following the removal of the initiation M et residue of yeast R pt1, the N ‐terminal P ro residue is either unmodified, mono‐methylated, or di‐methylated, and that this N ‐methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the P ro‐ L ys ( PK ) sequence at positions 3 and 4 of yeast R pt1, we generated mutant strains expressing an R pt1 allele that lacks this sequence. The absence of the PK sequence abolished N ‐methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N ‐methylation of R pt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast.