Little is known regarding the biological roles of archaeal proteases. The haloarchaeon Haloferax volcanii is an ideal model for understanding these enzymes, as it is one of few archaea with an established genetic system. In this report, a series of H. volcanii mutant strains with markerless and/or conditional knockouts in each known proteasome gene was systematically generated and characterized. This included single and double knockouts of genes encoding the 20S core ␣1 (psmA), ␤ (psmB), and ␣2 (psmC) subunits as well as genes (panA and panB) encoding proteasome-activating nucleotidase (PAN) proteins closely related to the regulatory particle triple-A ATPases (Rpt) of eukaryotic 26S proteasomes. Our results demonstrate that 20S proteasomes are required for growth. Although synthesis of 20S proteasomes containing either ␣1 or ␣2 could be separately abolished via gene knockout with little to no impact on growth, conditional depletion of either ␤ alone or ␣1 and ␣2 together rendered the cells inviable. In contrast, the PAN proteins were not essential based on the robust growth of the panA panB double knockout strain. Deletion of genes encoding either ␣1 or PanA did, however, render cells more sensitive to growth on organic versus inorganic nitrogen sources and hypo-osmotic stress and limited growth in the presence of L-canavanine. Abolishment of ␣1 synthesis also had a severe impact on the ability of cells to withstand thermal stress. This contrasted with what was seen for panA knockouts, which displayed enhanced thermotolerance. Together, these results provide new and important insight into the biological role of proteasomes in archaea.Detailed three-dimensional structures of archaeal proteasomes have provided a firm foundation for understanding how these elaborate nanocompartmentalized complexes mediate protein degradation (3, 11). While their thermostable properties make archaeal proteasomes ideal for structural studies, the optimum growth requirements of archaea, which often include extreme pH, salt, and/or temperature, have limited our fundamental knowledge of how these proteolytic enzymes work in the ubiquitin-free archaeal cell.Halophilic archaea or haloarchaea have developed into model organisms that are used to study many biological processes. Recent advances in haloarchaeal genome sequencing (4,6,14,15) and the development of new genetic tools, such as the targeted and markerless deletion of chromosomal genes (1), have made this group of microbes ideal for providing insight into cell physiology.Haloferax volcanii is a haloarchaeon which encodes at least five protein components associated with the proteasome system. These include the ␣1, ␤, and ␣2 proteins, encoded by psmA, psmB, and psmC, respectively, which form at least two 20S proteasome subtypes of differing subunit compositions (␣1␤ and ␣1␣2␤) (8,20). H. volcanii also encodes two proteasome-activating nucleotidase (PAN) proteins, PanA (panA) and PanB (panB), closely related to the regulatory particle triple A-ATPase (Rpt) proteins of eukaryal 26S pro...

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