Nα-Acetylation and Proteolytic Activity of the Yeast 20 S Proteasome*

Nα-Acetylation, catalyzed co-translationally with Nα-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3. The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants,nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the α1, α2, α3, α4, α7, and β3 subunits were acetylated with NAT1, the α5 and α6 subunits were acetylated with MAK3, and the β4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the α5 and α6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were Nα-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activitiesin vitro, suggesting thatNα-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1mutant than in the normal strain.