INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Energy-dependent proteolysis is not only vital to elimination of defective cellular proteins but is also central to regulation of cell division, metabolism, transcription, and other essential cellular functions (12, 22). A small group of related energy-dependent proteases have been identified from several diverse organisms; these proteases include Lon, FtsH (HflB), ClpAP, ClpXP, HslUV (ClpYQ), and the 26S proteasome (4, 39). Although this group of proteins shares limited primary sequence identity, the proteases have converged to a universal structure in which relatively nonspecific proteolytic active sites are compartmentalized from the cell by narrow openings (37, 67). The current model is that the degradation of biological substrates by this group of proteases requires additional energy-dependent proteins or protein domains for the recognition and/or unfolding of substrate proteins (21, 29, 65). Some of these energy-dependent components may not only participate in proteolysis but also have independent roles as chaperones (60). Thus, the energy-dependent component of these proteases may provide a proofreading step following the initial binding of protein substrates and enable the cell to distinguish between proteins destined for refolding, disaggregation, or destruction (23).

Some energy-dependent proteases are organized in a symmetry mismatch, including ClpAP and ClpXP which are complexes of the hexameric ClpA and ClpX ATPases interfaced with the ClpP protease of two heptameric rings (5). It is postulated that hydrolysis of nucleotides by the energy-dependent component results in rotation at the ATPase-protease interface (5) and that this rotation facilitates the processive degradation of substrate proteins (63). This mismatch, however, does not appear to be universal, since the fully functional Lon and FtsH proteases assemble into homomultimeric complexes with the ATPase and proteolytic components encoded by a single gene (10, 47).

Energy-dependent proteases, including Lon and proteasome complexes, are predicted from the genome sequences of archaea (8, 32, 34, 57). Little is known, however, about the biochemistry or biological significance of these energy-dependent proteolytic pathways in this unusual group of organisms. 20S proteasomes have been purified from diverse archaea (3, 13, 42, 74) and appear to have a self-compartmentalized structure which requires polypeptides to be at least partially unfolded prior to hydrolysis (72). This suggests that the archaeal 20S component alone, as purified, has little biological significance in native protein hydrolysis and suggests that additional components are necessary for protein degradation at the optimal growth temperature of these organisms. The related eucaryal 20S proteasome is fully functional in degrading proteins but only when associated with an energy-dependent 19S cap regulatory complex or an eight-subunit derivative of this complex, designated the base domain (21). Six of these base domain subunits are ATPases which are members of the AAA family (named AAA for ATPases associated with a variety of cellular activities) (46) and have recently been named Rpt proteins (for regulatory particle triple-A) (16). It is postulated that these energy-dependent subunits are responsible for the unfolding and/or translocation of substrate proteins into the central proteolytic chamber of the 20S proteasome.

Closely related homologs of the eucaryal ATPase proteasome subunits are predicted from the genome sequences of the archaea (8, 32, 34, 57). One homolog from Methanococcus jannaschii (MJ1176) has recently been purified from recombinant Escherichia coli and named PAN for proteasome-activating nucleotidase (84). This protein was purified as a 650-kDa complex of an N-terminal polyhistidine-tagged form of PAN in association with a derivative of PAN which had a deletion of residues 1 to 73 [PAN(1-73)]. The purified PAN complex had ATPase activity and activated the energy-dependent degradation of proteins by 20S proteasomes from Methanosarcina thermophila and Thermoplasma acidophilum by four- to ninefold (84). Characteristic of AAA proteins, the deduced sequence of PAN reveals a highly conserved Walker A motif (211-GPPGTGKT-218) which is predicted to be involved in coordination of Mg²⁺ and formation of hydrogen bonds with nucleotide triphosphates including the - and -phosphates (54, 66). A modified Walker B motif or "DEAD" box which is also predicted to be involved in Mg²⁺ binding and ATP hydrolysis is conserved and includes residues 270-DEID-273 of PAN (9). Additionally, a SRH or second region of homology motif [(T/S)-(N/S)-X₅-D-X-A-X₂-R-X₂-R-X-(D/E)] which distinguishes AAA proteins from the broader family of Walker-type ATPases is also conserved and spans residues 315 to 333 of PAN. The SRH motif appears to be important in ATP hydrolysis but not ATP binding (31). The PAN sequence also has a highly charged N-terminal coiled-coil spanning residues 49 to 83 which is conserved in other AAA proteins (84). This coiled-coil may play an important role in regulating nucleotide hydrolysis, protein-protein interaction, and/or other activities as proposed for other AAA proteins including the ATPase subunits of the 26S proteasome as well as ClpA and ClpB (38, 51, 56, 68).

In this communication, the biochemical and physical properties of the 20S proteasome and PAN proteins of M. jannaschii are presented. Our results demonstrate that this 20S proteasome requires a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins. The N-terminal coiled-coil region of PAN is not required for this reaction and is not needed for association of the PAN protein into an ~12-subunit complex. However, deletion of the N-terminal 73 residues does appear to influence the biochemical properties of nucleotide hydrolysis mediated by the PAN protein.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Materials. Biochemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Other organic and inorganic chemicals were from Fisher Scientific (Atlanta, Ga.) and were analytical grade. Restriction endonucleases and DNA-modifying enzymes were from New England BioLabs (Beverly, Mass.) or Promega (Madison, Wis.). Oligonucleotides were from Genemed Synthesis (San Francisco, Calif.). Polyvinylidene difluoride membranes were from MicroSeparations (Westborough, Mass.). The M. thermophila and T. acidophilum 20S proteasomes were purified as previously described (41, 55).

Strains and media. Escherichia coli strains included TB-1 F ara (lac-proAB) rpsL (Str^r) [80dlac(lacZ)M15] thi hsdR(r_K m_K⁺) (New England BioLabs) and BL21 (DE3) F ompT [lon] hsdS_B(r_B m_B) (an E. coli B strain) with DE3, a  prophage carrying the T7 RNA polymerase gene (59). Strains were grown in Luria-Bertani (LB) medium or LB medium supplemented with 50 mg of spectinomycin per liter, 100 mg of ampicillin per liter, and/or 50 mg of kanamycin per liter as needed. M. jannaschii strain JAL-1 was grown to mid-log phase with H₂ and CO₂ by the method of Boone et al. (6).

Enzyme assays. Protein concentrations were determined by the bicinchoninic acid method (58) (Pierce, Rockford, Ill.) for the PAN proteins and by the Coomassie blue dye-binding method (7) (Bio-Rad, Hercules, Calif.) for the 20S proteasome, using bovine serum albumin as the standard. Peptide-hydrolyzing activity was assayed by the release of 7-amino-4-methylcoumarin (fluorescence) or -naphthylamine (absorbance) as previously described (41, 42, 74). Protein hydrolysis was monitored as the generation of -amino groups using fluorescamine as previously described (74) with methionine as a standard. The release of inorganic phosphate (P_i) was measured using malachite green with modifications as previously described (35, 36). For analysis of various nucleotide-hydrolyzing activities, enzyme (2 µg per ml) was incubated with 1 mM nucleotide in 25 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] at pH 8.0 with 100 mM NaCl, 10 mM MgCl₂ (TES-NaCl-MgCl₂ buffer) at 80°C for PAN or 65°C for PAN(1-73). For determination of the kinetic parameters, enzyme (4 µg per ml) was incubated with ATP or CTP at 0.05, 0.1, 0.25, 0.5, and 1 mM using similar assay conditions. Initial velocity was determined by measuring the release of P_i in triplicate using four time points from 0 to 6 min. Initial velocity was plotted as a function of substrate concentration by the Lineweaver-Burk method (11). Similar results were obtained using Michaelis-Menten and Eadie-Hofstee plots (11). The effects of nucleotide diphosphates were measured by preincubating the enzyme (2 µg per ml) with or without 1 mM CDP or ADP for 15 min in TES-NaCl-MgCl₂ buffer at 21°C before addition of 1 mM ATP or CTP and then assayed as described above. The pH optimum of enzymes was measured using the following 25 mM buffers: 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5 to 7), TES (pH 7 to 8), N-tris(hydroxymethyl)methylglycine (Tricine) (pH 8 to 8.8), and 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) (pH 8.9 to 10).

Protein techniques. Molecular masses of purified protein subunits were estimated by reducing and denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% polyacrylamide gels which were then stained with Coomassie blue R-250. The molecular mass standards were phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) (Bio-Rad). N-terminal sequencing by Edman degradation was as previously described using purified proteins separated by SDS-PAGE (74).

Cloning and site-directed mutagenesis of the M. jannaschii proteasome genes. PCRs were performed using a Gene Cycler (Bio-Rad) to amplify DNA fragments containing M. jannaschii genes encoding the  and  subunits of the 20S proteasome and PAN protein. Oligonucleotide primers, template plasmid DNA, and cloning procedures are shown in Table 1. The DNA sequences of the fragments generated by PCR amplification were verified by sequence analysis of both strands of plasmid DNA (DNA Sequencing Facility, Department of Microbiology and Cell Science, University of Florida, Gainesville).

Expression of M. jannaschii genes in E. coli. The M. jannaschii genes encoding the subunits of the 20S proteasome and PAN protein were expressed separately in E. coli BL21(DE3) using the bacteriophage T7 RNA polymerase-promoter system and plasmids listed in Table 1, as previously described (41, 59) (Novagen, Madison, Wis.). An E. coli host strain containing the ileX and argU genes, encoding tRNA_AUA and tRNA_AGA/AGG, on plasmid pSJS1240 (33) was used to maximize the overall yield of M. jannaschii protein.

Purification of recombinant 20S proteasome and  and (pro) proteins. E. coli(pMj or pMjpro) cells were thawed in 6 volumes (wt/vol) of 10 mM sodium phosphate buffer at pH 7.2 containing 1 mM dithiothreitol (DTT) and passed through a French pressure cell at 20,000 lb/in². This was followed by centrifugation at 16,000 × g for 30 min at 4°C. For purification of the 20S proteasome, cell lysates containing the  and (pro) proteins were mixed at a 1:1 stoichiometry to a final concentration of ~10 mg of protein per ml, heated to 85°C for 15 min, and then chilled to 0°C for 30 min. The heated sample was centrifuged at 15,000 × g for 30 min at 4°C, which removed the majority of E. coli protein contaminants. The supernatant at 2.4 mg of protein per ml was concentrated by dialysis against PEG 8000 in bovine serum albumin (BSA)-treated dialysis tubing (cutoff, 3.5 kDa) (Pierce) to ~13 mg of protein per ml. The sample (50 mg) was added to 10 ml hydroxyapatite (Bio-Rad) equilibrated with 10 mM sodium phosphate buffer at pH 7.2 containing 5 mM DTT. Contaminating proteins were removed with 250 mM sodium phosphate, and samples which hydrolyzed N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-Amc) were obtained after elution with 500 to 750 mM sodium phosphate buffer at pH 7.2 containing 5 mM DTT. These fractions were dialyzed against 20 mM sodium phosphate buffer at pH 7.2 with 5 mM DTT for 18 h at 4°C and centrifuged at 15,000 × g for 30 min at 4°C. The protein was concentrated as described above to a final concentration of 6 to 10 mg of protein per ml. The proteasome was further purified after gel filtration in a Superose 6 HR 10/30 column (2.5 by 28.5 cm) (Pharmacia) equilibrated with 20 mM Tris buffer at pH 7.2 with 150 mM NaCl, 5 mM DTT, and 10% glycerol. The 700-kDa fractions hydrolyzing Suc-LLVY-Amc were collected and determined to be pure by reducing SDS-PAGE as well as by transmission electron microscopy to ensure that E. coli membrane vesicles were not contaminating the 20S proteasome sample. For purification of the  and (pro) proteins, cell lysate containing the individual proteins was heated, centrifuged, concentrated, and applied to a Superose 6 column as described above.

Purification of recombinant PAN proteins. For purification of N-terminal six-histidine-tagged PAN (His₆-PAN), E. coli(pMjPAN5) cells were thawed in 6 volumes (wt/vol) of 20 mM Tris buffer at pH 7.9 containing 5 mM imidazole and passed through a French pressure cell at 20,000 lb/in². This was followed by centrifugation at 16,000 × g for 30 min at 4°C. Sample was applied to a Ni²⁺-Sepharose column (4.8 by 0.8 cm) (Pharmacia) equilibrated with 20 mM Tris buffer at pH 7.9 containing 5 mM imidazole and then washed with 10 ml of 20 mM Tris buffer at pH 7.9 containing 60 mM imidazole. The His₆-PAN protein was eluted with 10 ml of 20 mM Tris buffer at pH 7.9 containing 500 mM imidazole, pooled, and dialyzed against 25 mM Tris buffer at pH 7.5. Fractions with ATP-hydrolyzing activity at 65°C were pooled and determined to be pure by reducing SDS-PAGE.

Production of antibodies against PAN(1-73) and 20S proteasome proteins. The purified recombinant PAN(1-73) protein of M. jannaschii and the  and  proteins of the M. thermophila 20S proteasome (41) were applied to a reducing SDS-12% polyacrylamide gel. Gel fragments containing the individual proteins (300 µg) were used to generate polyclonal antibodies in rabbits with Freund's adjuvant according to the supplier (Cocalico Biologicals, Reamstown, Pa.).

Purification and analysis of PAN and 20S proteasome proteins from M. jannaschii. M. jannaschii cultures were chilled to 0°C, cells were harvested aerobically by centrifugation at 5,000 × g for 15 min at 4°C, and cell material (0.5 g per liter) was stored at 70°C. Cells were thawed in 17 volumes (wt/vol) of 50 mM Tris buffer at pH 8.0 with 150 mM NaCl, 10 mM MgCl₂, 1 mM ATP, 10 mM -mercaptoethanol, 10% glycerol, and 0.1% Triton X-100. The cell suspension was passed through a French pressure cell twice at 10,000 lb/in² and then centrifuged at 15,000 × g for 30 min at 4°C. Cell lysate was applied to a Superose 6 HR 10/30 column equilibrated with Tris pH 8.0 buffer with 150 mM NaCl and 10% glycerol followed by ion-exchange chromatography in a MonoQ HR 5/5 column equilibrated with Tris pH 8.0 buffer with 100 mM NaCl and 10% glycerol which was then developed with a linear NaCl gradient (0.1 to 1 M NaCl in 15 ml of Tris pH 8.0 buffer). In some experiments, ion-exchange chromatography preceded gel filtration. Protein fractions were analyzed for NEM-inhibitable hydrolysis of ATP as described above and analyzed by immunoblotting after denaturing and reducing SDS-12% PAGE (27). PAN complex was also analyzed by immunoblotting of proteins separated by nondenaturing gel electrophoresis and capillary transferred to polyvinylidene difluoride membranes using 10 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.0 with 20% methanol. Primary antibodies as described above and goat anti-rabbit immunoglobulin G (IgG) linked to alkaline phosphatase were used for detection (Southern Biotechnology Associates, Birmingham, Ala.).

Electron microscopy of 20S proteasome and PAN proteins. Recombinant M. jannaschii PAN or PAN(1-73) complexes and the 20S proteasome were incubated separately and in equimolar ratios at 21 or 50°C for 15 min in TES-NaCl-MgCl₂ buffer with 1 mM ATP. Proteins were placed on 200-mesh grids coated with Formvar or carbon film, floated on water to remove salts, fixed with 2% cacodylate-buffered glutaraldehyde, briefly stained with 1% aqueous uranyl acetate, and treated with 0.01% bacitracin to improve spreading as previously described (74). Samples were viewed and photographed on a Zeiss EM-10CA transmission electron microscope operated at 80 kV. Stereo pairs were taken at an original magnification of ×100,000 using the Zeiss goniometer stage tilted at ±10°.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Translation limits heterologous synthesis of M. jannaschii 20S proteasome and proteasome-activating nucleotidase (PAN) proteins. Three open reading frames (ORFs) which code for putative proteins involved in energy-dependent protein degradation were identified from the genome sequence of the methanoarchaeon M. jannaschii (8). These ORFs include MJ0591 and MJ1237, which are predicted to encode the  and  subunits of a 20S proteasome as well as MJ1176 which has similarity to the ATPase (Rpt) subunits of the eucaryal 26S proteasome (8, 84). To further investigate archaeal proteasome-mediated protein degradation, these ORFs were separately cloned and expressed in E. coli. When an E. coli host strain with wild-type levels of tRNA was used, the yield of recombinant M. jannaschii protein was less than 0.5% of the total cell protein as determined by SDS-PAGE and immunoblotting. Analysis of the codon composition of the three ORFs revealed that up to 12% of the codons required either tRNA_AUA or tRNA_AGA/AGG for translation, which are rare in E. coli. Therefore, a compatible plasmid (pSJS1240) (Table 1) with the E. coli ileX and argU genes encoding these rare tRNAs was introduced into the host (33). In the presence of plasmid pSJS1240, the overall yield of M. jannaschii protein was increased to as much as 10% of total soluble protein, confirming that the rare tRNAs limit the high-level production of these proteins in E. coli.

Molecular masses of recombinant 20S proteasome subunits are identical to those produced in vivo. The purified  and (pro) proteins of the M. jannaschii 20S proteasome, synthesized in recombinant E. coli, migrated as 34- and 29-kDa proteins as determined by reducing SDS-PAGE (Fig. 1A). The (pro) protein was produced from a derivative of MJ1237 which no longer codes for the putative  propeptide which spans amino acid residues 2 to 6. Whether the N-formyl-Met of the M. jannaschii (pro) is removed by the aminopeptidase of E. coli to expose a putative active-site Thr, residue 7 of the full-length  protein, remains to be determined. The molecular masses calculated from the ORFs encoding the  and (pro) proteins are each 5 kDa less than those estimated by SDS-PAGE. We are unaware of any covalent modifications occurring in E. coli which would increase the molecular mass of proteins by 5 kDa. Therefore, it is more likely that the 20S proteasome proteins from this hyperthermophile are not fully unfolded after boiling for 15 min in the presence of 0.6 M -mercaptoethanol and 2% SDS, and the partially folded structure is retarding the migration of the  and (pro) proteins during separation compared to that of the proteins used to estimate their molecular mass. Immunoblot analysis of M. jannaschii cell lysate separated by SDS-PAGE revealed that both subunits of the 20S proteasome are produced in this archaeon and are the same molecular mass as those purified from recombinant E. coli. Therefore, MJ0591 and MJ1237 have been designated the psmA and psmB genes (for proteasome A and B), which is consistent with the nomenclature of the related genes from M. thermophila (42).

View larger version (95K): [in this window] [in a new window]   FIG. 1.   M. jannaschii 20S proteasome proteins. (A) Proteins (2 µg per lane) purified from recombinant E. coli, separated by reducing SDS-12% PAGE, and stained with Coomassie blue. Lanes: 1, (pro) protein; 2,  protein; 3, 20S proteasome of 700 kDa; 4, proteasome fraction of ~1.5 MDa. The positions of molecular mass standards are indicated to the left of the gel. (B to E) Transmission electron micrographs of the proteasome proteins. (B) End-on rings (single arrowhead) and sidewise paired rings (double arrowheads) of the  protein. (C) Proteasome fraction of ~1.5 MDa composed of equimolar ratios of  and (pro) proteins (arrowhead) which purify with membrane vesicles (arrow). (D) Side views of views of 20S proteasomes of 700 kDa with arrowheads indicating the four stacked rings of the cylindrical particle. (E) Stereo pair depicting a multilevel symmetrical array of the 20S proteasomes of 700 kDa after incubation at 65°C in Tris buffer at pH 7.2 with 150 mM NaCl, 1 mM ATP, and 10 mM MgCl2. Protein arrays were not observed under similar conditions in the absence of 150 mM NaCl or heating to 65°C. Bar, 20 nm.

Assembly of the  and (pro) proteins of the 20S proteasome. The individual  and (pro) proteins of the M. jannaschii 20S proteasome did not hydrolyze the peptide substrate Suc-LLVY-Amc (Table 2). The (pro) protein formed low-molecular-mass complexes of monomers to trimers, as estimated by nondenaturing electrophoresis and gel filtration, which were not visible by electron microscopy (data not shown). This is analogous to the related  subunits of the 20S proteasomes of M. thermophila, T. acidophilum, and the eubacterium Rhodococcus erythropolis which do not self-assemble into distinct complexes and remain inactive even in the absence of the propeptide (41, 55, 81, 83). The M. jannaschii  protein, in contrast, appeared to form cylinders of double stacked protein discs of 10-nm diameter which did not have visible central channels (Fig. 1B). This spontaneous self-assembly has been observed for other archaeal and eucaryal -type proteins, including the  proteins of M. thermophila and T. acidophilum as well as the human 7 (HsC8) and Trypanosoma brucei 5 proteins which spontaneously form single, double, and/or four-stacked protein rings when produced in E. coli (19, 20, 41, 78, 83). Self-assembly of the ₁ and ₂ proteins of the eubacterium R. erythropolis, however, depends on the presence of -type proteins and suggests distinct differences in 20S proteasome assembly among these organisms (81).

View this table: [in this window] [in a new window]   TABLE 2.   Chymotrypsin-like activities of the purified M. jannaschii proteasomes and in vitro assembly of the  and (pro) proteins

Biophysical properties of the 20S proteasome complex. An active M. jannaschii proteasome of 700 kDa was purified from the assembly and contains equimolar ratios of the  and (pro) proteins based on SDS-PAGE (Fig. 1A) (Table 2). Electron microscopy of the proteasome reveals a four-stacked ring structure of 12 by 17 nm with a central channel which has structural similarities to other 20S proteasomes (Fig. 1D) (3, 14, 41, 45, 62, 74). Based on analogy to the T. acidophilum 20S proteasome (25), it is likely that the  protein of M. jannaschii forms the outer protein rings of the cylinder as well as the distinct central openings. If so, assembly of the  disc-like complexes with the (pro) protein may involve conformational changes in  which generate the central portal and 2-nm increase in diameter of the 20S proteasome (Fig. 1B and E). An additional fraction of at least 1.5 MDa which was active in peptide hydrolysis was purified from the assembly mixture and contained equimolar ratios of the  and (pro) proteins as well as vesicles (Fig. 1A and C). Whether two 700-kDa proteasomes associate into this larger complex and are not efficiently separated from vesicles or whether single 700-kDa proteasome particles associate directly with vesicles remains to be determined. However, the ~1.5-MDa proteasome fraction was 1.3-fold more active in Suc-LLVY-Amc hydrolysis than the 700-kDa proteasomes (Table 2). These results suggest that changes in protein conformation may enhance peptide-hydrolyzing activities of the 20S proteasome. Incubation of the 700-kDa proteasomes in the presence of buffer with 150 mM NaCl at 65°C resulted in the formation of symmetrical arrays of the 20S proteasome in end-on views as observed on electron micrographs (Fig. 1E). Stereo pairs of these arrays (Fig. 1E) reveal that the proteasomes are not in a planar arrangement but instead are multilevel. It is likely that heating the proteins in the presence of salt promotes ionic and/or hydrophobic interactions between the outer walls of the 20S cylinder. Using similar methods, the spontaneous formation of arrays has not been observed for the 20S proteasome of M. thermophila and, thus, does not appear to be a universal feature of these proteins (40).

Multisubstrate and proteolytic activities of the 20S proteasome. The M. jannaschii 20S proteasome degraded -casein (Fig. 2). The rate of protein hydrolysis increased linearly to at least 100°C (Fig. 2) and was not influenced by ATP (data not shown). The highest rate of -casein hydrolysis measured was 1.5 and 3.2 times faster than that of the 20S proteasomes of T. acidophilum and M. thermophila (Fig. 2). To our knowledge, this is the highest reported rate of protein hydrolysis catalyzed by a 20S proteasome (1, 13, 41, 74). At the extreme assay temperatures used in this experiment, it is likely that -casein is thermally denatured, and additional proteins such as chaperones are not needed to unfold this polypeptide for entry into the central proteolytic chamber of the 20S proteasome.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Effect of temperature on -casein degradation by archaeal 20S proteasomes. Hydrolysis of bovine -casein was measured at the temperatures indicated using 0.2 mg of substrate protein and 0.05 mg of purified 20S proteasome in 1 ml of 20 mM Tris-1 mM DTT (pH 7.2). Abbreviations: Mj20S, Ta20S, and Mt20S, M. jannaschii, T. acidophilum, and M. thermophila 20S proteasomes, respectively; grps, groups.

Purification of the proteasome-activating nucleotidase (PAN). Transcription of the putative pan gene in E. coli resulted in the synthesis of three proteins of 47, 39, and 30 kDa which were not apparent in the absence of the coding sequence for this protein. This suggested that either the protein produced from the M. jannaschii gene induced the overproduction of E. coli stress response proteins or recombinant PAN proteins of variable molecular mass were generated. The 47- and 39-kDa proteins were stable after heating the cell lysate to 85°C and copurified after gel filtration as a 600-kDa fraction which hydrolyzed ATP at a rate of 1.8 µmol of P_i per min per mg of protein at 65°C (Fig. 3, lane 1). Protein sequence analysis revealed that the first 10 residues of the 47-kDa protein were identical to residues 2 to 11 of the PAN protein sequence deduced from DNA (Fig. 4). The first 10 residues of the 39-kDa subunit were found to be identical to residues 74 to 83 of the predicted PAN protein (Fig. 4). This suggests that there is an internal ribosome binding site recognized by E. coli within the full-length mRNA which accounts for the production of the shorter 39-kDa protein in E. coli. In addition, it is likely that the aminopeptidase of E. coli cleaved the N-terminal methionine residue of the full-length PAN protein; however, the N-terminal methionine of the 39-kDa PAN was not accessible for cleavage by the host peptidase. Using an alternative approach, the coding sequence for PAN was positioned in frame to produce full-length PAN with an N-terminal six-histidine tag (His₆-PAN). Similar to the study by Zwickl et al. (84), the 39-kDa PAN copurified with the full-length His₆-PAN after Ni²⁺ chromatography (Fig. 3, lane 2). However, in our study, the 39-kDa PAN appeared to copurify at an equimolar ratio with the full-length His₆-PAN compared to a much lower ratio for the previously purified complex (84). The His₆-PAN complex, of our studies, hydrolyzed ATP at a rate which was over 60% less than that of the untagged PAN complex described above. In addition, the His₆-PAN complex was less stable than the untagged PAN complex after heating to 85°C or long-term storage at 4°C. These results suggest that the N-terminal His₆ tag may influence PAN stability and activity.

View larger version (82K): [in this window] [in a new window]   FIG. 3.   M. jannaschii PAN proteins from recombinant E. coli. Protein (2 µg) was separated by reducing SDS-12% PAGE and stained with Coomassie blue. Lanes: 1, full-length PAN and PAN(1-73) from recombinant E. coli/pMjPAN2; 2, His6-tagged PAN and PAN(1-73) from recombinant E. coli/pMjPAN5; 3, PAN(1-73) from recombinant E. coli/pMjPAN8; 4, full-length PAN from recombinant E. coli/pMjPAN7; 5, molecular mass standards with the masses indicated to the right of the gel.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Site-directed mutagenesis of the Shine-Dalgarno site located within the M. jannaschii pan gene recognized in E. coli. Capitalized nucleotides are from plasmid pET24b, and lowercase nucleotides are from the pan gene. The Shine-Dalgarno site of pET24b used for synthesis of the full-length PAN protein in recombinant E. coli is double underlined. The nucleotides targeted and the oligonucleotide (Oligo) used for site-directed mutagenesis are highlighted and indicated above the DNA coding region, respectively. Amino acid residues identical to the N-terminal protein sequence determined by Edman degradation of the purified 47-kDa PAN are underlined, whereas those of the purified 39-kDa PAN(1-73) are underlined and italicized. Amino acid positions of the PAN protein deduced from the chromosomal DNA are indicated on the right.

PAN produced in M. jannaschii. Immunoblot analysis revealed that only the PAN protein of 47 kDa is detectable in M. jannaschii cells when grown on H₂ and CO₂ at 65°C (Fig. 5). These results demonstrate that the pan gene is expressed in M. jannaschii and generates the full-length protein. Partial purification of PAN from M. jannaschii cell lysate reveals that the protein is associated in a distinct 550-kDa complex as determined by gel filtration, electrophoresis, and immunoblot analysis (data not shown). It remains to be determined if the 550-kDa PAN complex purified from M. jannaschii is a homooligomer or associates with additional proteins.

View larger version (50K): [in this window] [in a new window]   FIG. 5.   PAN produced in M. jannaschii. Proteins were separated by reducing SDS-12% PAGE and analyzed by Western blot using anti-PAN(1-73) antibodies. Lanes: 1, total protein (1 to 2 µg) produced in recombinant E. coli/pMjPAN2 which synthesizes PAN proteins of 47, 39, and 30 kDa; 2, M. jannaschii cell lysate (20 µg) which produces only the full-length PAN of 47 kDa.

Biophysical properties of recombinant PAN. The full-length PAN and PAN(1-73) proteins synthesized in recombinant E. coli each form homooligomeric complexes of 550 and 500 kDa, respectively, as determined by gel filtration. The results suggest that PAN alone is able to form a complex of ~12 subunits and does not require the first 73 amino acids for this association. The PAN complexes were highly stable in the absence of ATP which contrasts with related proteins such as ClpA, ClpX, and NEM-sensitive fusion protein (NSF) which require nucleotide binding for complex stability (24, 26, 43). The PAN proteins were also stable after heating to 85°C in polypropylene tubes; however, they were readily inactivated when incubated in glass tubes much like ClpX (75). Nondenaturing PAGE reveals that each PAN complex migrated as a distinct band, which was in good agreement with its estimated high molecular mass (Fig. 6). The separated band of PAN protein catalyzed ATP hydrolysis and was inhibited by NEM, as determined by in-gel staining for inorganic phosphate (Fig. 6). A significant fraction of PAN(1-73), however, did not enter the gel, which suggests that it is somewhat prone to aggregation under the electrophoretic conditions used (Fig. 6). When PAN(1-73) is maintained in optimal buffer and salt conditions, however, the protein does not aggregate based on gel filtration chromatography (data not shown). Preincubation of either PAN protein with NEM at 65°C prior to electrophoresis did not influence their migration which contrasts with the related HslU of E. coli which dissociates into monomers in the presence of NEM (79).

View larger version (57K): [in this window] [in a new window]   FIG. 6.   Native gel separation of M. jannaschii PAN proteins. Lanes: 1, apoferritin of 443 kDa; 2, thyroglobulin of 669 kDa; 3, PAN(1-73) of 500 kDa based on gel filtration; 4 to 6, full-length PAN of 550 kDa based on gel filtration. The proteins were stained with Coomassie blue (lanes 1 to 4) or stained for the generation of Pi from ATP in the absence (lane 5) or presence (lane 6) of 10 mM NEM.

View larger version (108K): [in this window] [in a new window]   FIG. 7.   Transmission electron micrographs of M. jannaschii PAN. The arrowhead in panel A indicates side view in which PAN resembles a comma-shaped complex. The arrowhead in panel B points to a more face-on view in which the head of the comma is at top. Distinct individual subunits are seen as white globular components of each assembly. Larger clusters probably represent random associations created when sample was dried for transmission electron microscopy observation. Bars, 10 nm.

Biochemical properties of PAN. PAN(1-73) was used as a comparison to the full-length protein to examine the influence of the predicted N-terminal coiled-coil, which spans residues 49 to 83, on the biochemical properties of PAN. Both the full-length PAN and PAN(1-73) proteins have optimal ATPase activity at pH 7 to 8 and are more active at pH 8 to 10 (50 to 75% of the optimum) than at pH 5.5 (5 to 35% of the optimum). The ATPase activity of the full-length PAN protein is optimal at 80°C which is close to the optimal growth temperature of 85°C for M. jannaschii. In contrast, the ATPase activity of PAN(1-73) is optimal at 65°C. The ATPase activity of the recently characterized complex of His₆-PAN and PAN(1-73) is optimal at 73°C (84) which is between the optimal temperature for the PAN and PAN(1-73) complexes.

View this table: [in this window] [in a new window]   TABLE 4.   Effects of various compounds on the ATPase activity of the M. jannaschii PAN and PAN(1-73) proteins

View this table: [in this window] [in a new window]   TABLE 5.   Kinetic parameters of the M. jannaschii PAN and PAN(1-73)

PAN is required for ATP-dependent hydrolysis by archaeal 20S proteasomes. The PAN proteins were investigated for activation of protein hydrolysis mediated by archaeal 20S proteasomes. Both full-length PAN and PAN(1-73) complexes stimulated the degradation of -casein by the M. jannaschii 20S proteasome by 1.4 ± 0.04- to 2.2 ± 0.19-fold within 15 min, this stimulation was completely dependent on the presence of either ATP or CTP (data not shown). No significant energy-dependent hydrolysis of -casein was observed in the presence of ADP, and stimulation was optimal at molar ratios ranging from 0.5:1 to 4:1 of PAN and 20S proteasome complexes (data not shown). Furthermore, the PAN (Fig. 8) and PAN(1-73) complexes (data not shown) stimulated by 2- to 4-fold the degradation of -casein by the M. thermophila 20S proteasome in the presence of ATP, but not ADP. Under these assay conditions, the rate of ATP hydrolysis by PAN was not influenced by the addition of the synthetic peptide Suc-LLVY-Amc or the 20S proteasome alone but was enhanced by the addition of -casein by 1.22 ± 0.07-fold or -casein with the 20S proteasome by 1.38 ± 0.08-fold (data not shown). In addition, CTP substituted for ATP in stimulation of protein hydrolysis (data not shown), which is consistent with other energy-dependent proteases including the eucaryotic 26S proteasome (18), E. coli Lon (70), E. coli FtsH (64), and the complex of M. jannaschii His₆-PAN and PAN(1-73) with the T. acidophilum 20S proteasome (84).

View larger version (22K): [in this window] [in a new window]   FIG. 8.   -Casein degradation by the 20S proteasome is stimulated by PAN. PAN and M. thermophila 20S proteasome were mixed together in a 2:1 molar ratio in 25 mM TES buffer (pH 8) with 10 mM MgCl2, 1 mM nucleotide, and 2 mg of -casein per ml and incubated at 55°C. The amounts of -amino groups (grps) generated by protein hydrolysis were determined. Data presented are the averages of three separate experiments. Symbols: , PAN and 20S proteasome with ATP; , 20S proteasome with ATP; , PAN and 20S proteasome with ADP; , PAN and 20S proteasome with AMP-PNP.

PAN associates with archaeal 20S proteasomes. The purified M. jannaschii 20S proteasome and PAN proteins were mixed at equimolar ratios and then incubated in the presence of ATP and Mg²⁺ at 50°C. Electron micrographs of these mixtures reveal particles which resemble the 20S core capped either singly or doubly on each end by PAN protein complexes (Fig. 9). The singly capped proteasome-associated PAN complexes were ~24 nm long and 12 to 14 nm wide. These micrographs are consistent with the ability of the PAN proteins to stimulate proteolysis by 20S proteasomes and suggest that archaeal 20S proteasomes may interact with PAN to form a complex which resembles the 26S proteasome subcomplex recently described for S. cerevisiae (21). The efficiency of reconstitution of these complexes, however, was less than 0.5%, suggesting that the interaction between the proteins may be transitory and not stable during electron microscopy, additional factors are necessary for stable complex formation, and/or other possibilities yet to be determined.

View larger version (122K): [in this window] [in a new window]   FIG. 9.   Transmission electron micrographs of reconstituted 20S proteasome and PAN proteins. (A) Untilted view of particle generated during reconstitution of the PAN(1-73) and 20S proteasome. The arrowhead indicates cylindrical 20S proteasome assembly with apparent assemblies of PAN(1-73) at both ends. (B) Stereo view of a single 20S proteasome cylinder with a putative PAN assembly at the left end. (C) Stereo view of 20S proteasome arrays with indistinct patchy material on top of arrays in center and at left, which probably represent PAN complexes. Bars, 20 nm.