ABSTRACT Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N α acetylation of the initiator methionine of the α1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of α1 in H. volcanii cells. To further examine this, α1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP “gate” formed by the α1 N terminus were examined for their N α acetylation. Both the “gate” deletion and Q2A substitution completely altered the N α -acetylation pattern of α1, with the deletion rendering α1 unavailable for N α acetylation and the Q2A modification apparently enhancing cleavage of α1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two α1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although α1 proteins with Q2D, Q2P, and Q2T substitutions were N α acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of α1 as rings in N α -acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these α1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N α acetylation of α1 is important in CP assembly and activity, high levels of α1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP “gate” impairs the ability of cells to overcome salt stress.