The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its Nα Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N-alpha acetylation of the initiator methionine of the alpha 1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of alpha 1 in H. volcanii cells. To further examine this, alpha 1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP gate formed by the alpha 1 N terminus were examined for their N-alpha acetylation. Both the gate deletion and Q2A substitution completely altered the N-alpha-acetylation pattern of alpha 1, with the deletion rendering alpha 1 unavailable for N-alpha acetylation and the Q2A modification apparently enhancing cleavage of alpha 1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two alpha 1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although alpha 1 proteins with Q2D, Q2P, and Q2T substitutions were N-alpha acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of alpha 1 as rings in N(-alpha)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 alpha 1 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-alpha acetylation of alpha 1 is important in CP assembly and activity, high levels of alpha 1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP gate impairs the ability of cells to overcome salt stress.