Genetic and Proteomic Analyses of a Proteasome-Activating Nucleotidase A Mutant of the Haloarchaeon Haloferax volcanii

Abstract

The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.Phosphorylation is one of the most important and widespread posttranslational modifications of proteins. Its usefulness comes in the form of its strong perturbing forces, which modulate structure and function in a profoundly effective manner (27). Phosphorylation and dephosphorylation of proteins can occur quite rapidly, making this mechanism ideal for controlling adaptive responses to environmental cues and changing intracellular conditions. These appealing characteristics have made phosphorylation the modification of choice for regulating a number of vital cellular processes, many of which overlap between the branches of life. It is estimated that in eukaryotes, as much as 30% of the proteome is phosphoryla...

Proteasomal Components Required for Cell Growth and Stress Responses in the Haloarchaeon Haloferax volcanii

“…However, the ␣1 and/or PanA deficiencies did not significantly impact the overall cell yield of these strains (Table 2). In a previous study, we reported a ⌬panA strain (GG102) that displayed a reduced growth rate and cell yield compared to its parent, DS70, in ATCC 974 medium (9). ATCC 974 is a complex medium that contains a reduced level of sodium chloride, i.e., 2.1 M rather than to the 2.5 M present in YPC and GMM used in this study.…”

“…The reason for this finding remains to be determined. Our recent characterization of the (phospho)proteome of the related panA mutant strain GG102, which harbors a mevinolin resistance marker insertion and deletion within the panA gene, reveals not only a global increase in the number of different phosphoproteins but also an increase in the abundance of proteins related to stress responses as well as Cdc48-related triple-A ATPase homologs (9). Whether any of these proteins compensate in part for the loss of the PanA function and render the cell more resistant to thermal stress than wild-type cells remains speculative.…”

“…These mutant strains include one with a novobiocin resistance marker inserted into the coding region of the psmC gene, encoding the ␣2 protein (8), and a second strain with a deletion of ϳ100 bp and an insertion of a mevinolin resistance marker into the panA gene, encoding the Rpt-like PanA protein (9). The former strain (WFD11 [psmC1::gyrB*-gyrAЈ]) was used to purify 20S proteasomes of ␣1␤ composition, and the (phospho)proteome of the latter strain (GG102 [⌬panA::hmgA*]) was investigated by tandem mass spectrometry based the robust number of phosphoproteins detected by two-dimensional electrophoresis in its proteome compared to what is the case for the parent strain, DS70 (9). Together, these mutant strains have provided insight into proteasome structure and function, but they are limited in number and scope.…”

Proteasomes and protein conjugation across domains of life

“…Reporter gene constructs and proteomic methods have been used to detect the accumulation of proteins in proteasome-deficient archaeal cells 105,107–109 . According to these studies, the proteasome seems to be important for controlling the levels of proteins involved in key cellular processes and can destabilize proteins with hydro phobic C termini 105,107–109 .…”

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…First, the phosphoryl group of PϳEI from Salmonella enterica serovar Typhimurium can be transferred to the intermediately phosphorylated active-site residue of acetate kinase (23). Second, PϳEIIA of the dihydroxyacetone phosphorylation system of numerous bacteria and several archaea (24) transfers its phosphoryl group to an ADP molecule tightly bound to the active site of the dihydroxyacetone kinase subunit DhaL, which subsequently phosphorylates dihydroxyacetone bound to DhaK (18). Some enterobacteria possess a more complex dihydroxyacetone-specific multidomain protein composed of an EIIA-like domain and an HPr-like domain followed by a truncated EI-like domain containing the conserved phosphorylatable histidine.…”