The proteasomal ATPases’ C-termini contain a conserved HbYX motif
If the extreme C-termini of these ATPases are important for its association with the 20S, then the critical residues may be conserved, since the requirements for complex formation and gate opening by PAN and the 19S ATPases appear similar (Smith et al., 2005
). We therefore compared their C-terminal sequences in various archaea and eukaryotes (). While the terminal arginine residue in PAN is not conserved, the penultimate residue is a tyrosine in PAN from 9 different archaeal species (see Supplement Table S1
), although in five archaeal species, it is a phenylalanine. A penultimate tyrosine is also found in the four ATPases, Rpt1, Rpt2, Rpt3 and Rpt5 (but not Rpt4 and Rpt6) in 26S proteasomes from humans, rats, mice, drosophila, arabidopsis, nematodes (except Rpt1) and yeast. A hydrophobic residue precedes the penultimate tyrosine in PAN from all 14 archaea species and also in Rpt2, Rpt3, Rpt5, and Rpt6 of these eukaryotes. Thus the conserved C-terminal motif, HbYX, where Hb is a hydrophobic residue, Y is a tyrosine (or in some archaea, a phenylalanine), and X can vary widely, appears in nearly all known proteasomal ATPases. Interestingly, no such sequence is found in the C-termini of PA26 and PA28α, β or γ.
Figure 1 A) Carboxypeptidase treatment of the proteasomal ATPase regulatory complexes’ eliminates their ability to stimulate the 20S for peptide hydrolysis. A) Two residues preceding the C-terminal arginine in PAN are conserved in the eukaryotic 19S proteasome-regulatory (more ...)
PAN’s C-terminal residues are required to stimulate 20S gate opening
To determine if PAN requires its C-terminal arginine to associate with the 20S proteasome, PAN and the 20S were pretreated with carboxypeptidase B (CpB), which specifically removes basic C-terminal residues, and after addition of ATPγS, complex formation and gate opening in the 20S were assayed (Smith et al., 2005
). The stimulation of gate opening was monitored by measuring the hydrolysis of the quenched 9-residue fluorogenic substrate, LFP, whose entry into the 20S is very slow when the gate is closed (Smith et al., 2005
). Treatment with carboxypeptidase B did not affect the ability of the 20S proteasome to hydrolyze LFP and did not alter PAN’s ATPase activity or its ability to unfold GFP-ssrA (Supplement Fig. S1 a and b
). However, after carboxypeptidase B treatment, which should remove the arginine but not the penultimate tyrosine, PAN completely lost its ability to stimulate peptide entry (). By contrast, carboxypeptidase A, which cleaves primarily after hydrophobic residues, had no effect on the stimulation of peptide entry. Thus, PAN’s C-terminal arginine is required for gate opening, which can account for the recent finding that this residue is also required for the stimulation of casein degradation by PAN (Forster et al., 2005
Interestingly, if the PAN-20S complex was formed first by addition of ATPγS before the treatment with carboxypeptidase B, then PAN retained some ability to stimulate peptide hydrolysis (). Therefore, PAN’s C-terminal residues are essential for activation of gate opening and are accessible to carboxypeptidases when the ATPase complex is not associated with the 20S, but become less accessible once the ATPase-20S complex is formed. (It is noteworthy that this protection from carboxypeptidase inactivation is only partial, probably because the association between PAN and the 20S is transitory (Smith et al., 2005
). Thus, the C-terminus is likely to be located in the interface of the ATPase-20S complex, as shown below.
To investigate further the importance of PAN’s C-terminal arginine in gate-opening, we performed site-directed mutagenesis to remove it, replace it with other amino acids, or extend the C-terminus. When this arginine was deleted, PAN lost its ability to stimulate gate opening in the presence of ATPγS (as was found after treatment with carboxypeptidase B). However, mutating this arginine (R430) to alanine or tryptophan did not reduce PAN’s ability to stimulate the 20S, and mutation to a glycine (R430G) decreased it only slightly (). Because the R430G mutation still permitted gate opening, a side chain on the ultimate residue is not essential for 20S activation. Although many amino acids can replace this terminal arginine, surprisingly, replacement by a leucine markedly decreased PAN’s ability to stimulate the 20S, and replacement by an aspartate completely prevented activation. Furthermore, addition of an alanine C-terminal to the arginine blocked PAN’s ability to stimulate gate opening, even though an alanine in place of the arginine did not influence this activity. Thus, the binding site for the C-terminal sequences has some specificity, and while the ultimate residue does not require any specific side chain, basic side chains in this position do not allow function.
Mutagenesis to remove PAN’s C–terminal arginine destroys its ability to stimulate gate opening, but its replacement by certain other residues allows wild type activity
The penultimate HbY residues are essential for PAN to stimulate gate opening
To determine if the conserved hydrophobic and tyrosine residues in the HbYX motif are important for the association with the 20S and stimulation of gate opening, we systematically mutated these residues. Tyrosine Y429 was found to be absolutely essential for PAN’s activity (). When it was mutated to any of eight other residues, including hydrophobic, aromatic, or charged, none stimulated LFP hydrolysis. This lack of stimulation with a phenylalanine in this position was surprising since the penultimate residue in PAN from five archaeal species is a phenylalanine. Mutating the leucine preceding the tyrosine to any of 10 other amino acids confirmed that only hydrophobic (Hb) residues supported activity, but replacement by an arginine, aspartate, cysteine or proline prevented the stimulation of gate opening.
The penultimate Tyr-429 and preceding hydrophobic residue Leu-428 in PAN are required for 20S gate opening
The HbYX motif is required for PAN-20S complex formation
Mutations in the HbYX motif might block gate opening either by preventing the formation of the PAN-20S complex, or by allowing its formation but interfering with the gate-opening mechanism. We used electron microscopy as described previously (Smith et al 2005
) to test if the mutations that prevent activation also prevented complex formation. Five different PAN mutants were analyzed by EM. We observed extensive complex formation with those variants that stimulated gate opening, but did not observe any PAN-20S complexes with the PAN mutants that failed to stimulate gate opening (Table S2
). Thus, the failure of these PAN mutants to stimulate gate-opening is due to a failure to associate with the 20S.
Residues preceding the HbYX motif are not essential in gating
Although the residues preceding the HbYX motif were not conserved in other proteasomal ATPases, we mutated each of the four residues preceding the HbYX motif to alanines to determine their importance for activation of the 20S. None of these residues was absolutely essential for PAN’s stimulation of peptide hydrolysis, but some alanine replacements did reduce PAN’s activity. Potentially, this C-terminal sequence may adopt a helical conformation as was suggested for the C-termini of PA26 in the PA26-20S complex (Whitby et al., 2000
). To test this possibility, we inserted prolines at two positions in the C-termini to prevent helix formation or mutated the proline (P422) to an alanine (Table S3
). None of these mutations significantly reduced the stimulatory activity of PAN. Therefore, a C-terminal helix is probably not essential for formation of the PAN-20S complex or activation of gate opening. Also, insertion of four alanines before the seven C-terminal residues had little effect (), suggesting that these terminal residues are flexible or function relatively independently and fit into a constrained pocket that permits only certain residues.
PAN’s C-terminus moves from an aqueous to a hydrophobic environment upon complex formation
The susceptibility to carboxypeptidase B () suggested that PAN’s C-terminal residue is exposed in PAN, but not after association with the 20S. To analyze further the environment surrounding the C-terminal residues, we monitored tryptophan fluorescence. Since wild-type PAN and the Thermoplasma 20S proteasome both lack tryptophans, we analyzed the fluorescence spectra of mutants where a tryptophan was inserted in the C-terminal position (R430) or in place of the conserved hydrophobic residue (L428). Both mutations allowed wild-type activity ( and ). The maximum emission wavelength of the C-terminal tryptophan (W430) was 357nm (), which resembles that of free tryptophan in an aqueous solution. Accordingly, this fluorescence was quenched upon addition of acrylamide (not shown). Therefore, the ultimate C-terminal residue of PAN is indeed exposed to the solvent, where it appears available for docking with the 20S. However, when a tryptophan was placed two residues upstream (W427), it showed a λem max of 341nm, suggesting that this residue is located in a more hydrophobic environment.
Figure 2 The fluorescent spectrum of PAN R430W and PAN L428W mutants in solution and upon binding the 20S. A) Fluorescent emission spectra of the various PAN mutants. B) Fluorescent emission spectra of PAN R430W in the presence of 20S proteasomes without and with (more ...)
ATP-binding to PAN is essential for complex formation with the 20S (Smith et al., 2005
). One attractive model to explain this activation by ATP is that the C-terminal residues assume a more exposed conformation upon binding ATP, which promotes PAN-20S association, but not upon binding ADP, which inhibits complexation. Therefore, we tested whether the tryptophan at the 430 or 428 positions become more or less exposed to solvent upon binding ATP or ADP. However, we found no such change in the spectrum of PAN with either the solvent-exposed R430W or the more hydrophobicly exposed L428W after addition of ATP, ATPγS or ADP.
If the C-termini of PAN, like those of PA26 (Forster et al., 2005
), dock into pockets in the 20S’s a-ring upon complex formation, then the terminal tryptophan in the R430W PAN mutant should move from an aqueous to a more hydrophobic environment. After adding ATPγS to a mixture of PAN and proteasomes, the λem max
of this terminal tryptophan shifted from 357nm to 352nm, and the fluorescence intensity increased by over 50% (), as is characteristic of a tryptophan that shifts from an aqueous environment to a more hydrophobic one. By contrast, the addition of ADP did not change the λem max.
Also, addition of ATPγS to the 20S (which lacks tryptophans) did not change it’s background tyrosine fluorescence, and there was no change in the spectrum of the PAN L428W mutant upon association, since the λem max
of this tryptophan is already blue shifted (). At high concentrations, ADP competes with ATP and promotes PAN-20S dissociation (Smith et al., 2005
). Accordingly, if excess ADP was added after formation of the PAN-20S complex with ATPγS, then ADP shifted the λem max
to the red, and the fluorescence intensity decreased (not shown). Therefore, upon ATP-dependent association of PAN with the 20S, the C-termini move from an aqueous environment to a more hydrophobic one, presumably the pockets in the α-ring (see below).
PAN’s C-termini become polarized upon association with the 20S
Formation of the PAN-20S complex has been difficult to demonstrate because of its transient, and relatively weak association (Smith et al., 2005
). To quantitate the association of PAN with the 20S in solution, we analyzed the polarization of the C-terminal tryptophan in PAN R430W. Fluorescence polarization can be used to measure the rate of rotation of a fluorescent molecule, which can decrease upon association with another protein. When ATPγS (but not ADP) was added to a solution of PAN and the 20S, a 40 mP change in polarization was detected. Also, without the 20S present, W430 did not become more polarized upon addition of ATP, ATPγS or ADP. Thus, the terminal tryptophan becomes polarized upon association with the 20S. To determine what molar ratio of 20S to PAN gave maximal polarization, we added increasing amounts of 20S to a solution of PAN R430W and ATPγS. This assay is more quantitative than biochemical or EM approaches used previously to show complex formation, and because high concentrations of PAN and 20S could be used, stochiometric binding and saturation were demonstrated. Polarization approached maximal with two hexameric PAN complexes per 20S proteasome (), i.e. when most of the 20S was doubly capped with PAN, and most of the PAN was associated with a proteasome.
PAN binding to the 20S requires lysine-66 in its α-ring
Lysine 66 is located in the intersubunit pockets in the 20S’s α-ring, and when PA26 complexes with the Thermoplasma 20S, the C-terminal carboxyl group of PA26 forms a hydrogen bond with this lysine (Forster et al., 2005
). Accordingly, we found that PAN was unable to stimulate gate opening in K66A-20S mutant proteasomes, as assayed by measuring LFP hydrolysis (data not shown). Forster et. al. (2005)
had shown that this lysine is also required for the stimulation of casein degradation by PAN, which could be due to the requirement of Lys66 for gate-opening or for PAN-20S association. We therefore tested if K66 is also essential for the association of PAN and the 20S. In the presence of ATPγS, the K66A-20S, unlike wild-type proteasomes, did not alter the fluorescence spectrum of the terminal tryptophan in PAN-R430W () and did not cause its polarization (data not shown). These results indicate that PAN cannot stimulate gate opening in K66A-20S because it cannot associate with this particle and strongly suggest that PAN’s C-termini dock into the same intersubunit pockets as the C-termini of PA26.
Short peptides corresponding to PAN’s C-terminus induce 20S gate opening
Two possible models can explain the above results: 1) that insertion of PAN’s C-termini into the hydrophobic binding sites in the α-ring function by themselves like a “key-in-a-lock” to induce gate opening. 2) Alternatively, this interaction is necessary for PAN-20S complexation, but some other domain(s) in PAN (e.g. like the activation loop in PA28/26) are also necessary to induce the open-gate conformation. To test whether docking of PAN’s C-termini is sufficient to cause gate opening, we synthesized different length peptides that corresponded to PAN’s C-terminal sequence (), incubated them with proteasomes, and monitored gate opening by LFP hydrolysis. Surprisingly, peptides 7 to 10 residues long by themselves stimulated peptide degradation by 20S proteasomes by up to 9-fold. This large stimulation, which resembled the stimulation seen with PAN and ATP, was seen at the rather high peptide concentrations of 250 μM (). However, with the 7 residue peptide, near-maximal stimulation of peptide hydrolysis was evident at 10μM (See supplementary, Fig. S2
). These findings indicate rather high affinity binding of the 7-residue peptide, especially since in PAN, these C-termini are likely to function in a multimeric fashion and with a specific steric relationship. However, accurate determination of Ka’s are unreliable by this approach, because these C-terminal peptides (especially above 50μM), not only cause gate-opening, but also enter the 20S particle and compete at the active sites with LFP (whose hydrolysis is used to assay gate opening).
Figure 3 Peptides derived from PAN’s extreme C-terminal sequence induce gate opening and inhibit PAN-20S complex assembly. A) Ability of peptides of different lengths to stimulate gate opening. Peptides (250μM) were incubated with 0.2 μg (more ...)
The 7-residue peptide, HLDVLYR, stimulated gate opening much better than the 6-residue peptide, LDVLYR, and smaller peptides were ineffective. The small stimulation with the 6-residue peptide (up to 2-fold) reflects a requirement for peptide length rather than for an N-terminal histidine, since when this histidine was changed to an alanine in the peptide, it showed a similar ability to stimulate gate opening. Accordingly the corresponding mutation in full length PAN, A424H, stimulated gate opening like wild-type PAN. Surprisingly, the efficacy of the C-terminal peptides decreased at greater length, and the 10-residue peptide (EPAHLDVLYR) showed activity only above 200μM (data not shown). These findings indicate a steep dependence on peptide length. Presumably, a length of 7-residues is necessary for the peptide to occupy and assume a similar conformation as the C-termini of PAN in the intersubunit pockets.
Peptide-induced gate opening requires the HbYX motif and lysine-66 in the α-ring
Gate opening in the 20S can be activated non-specifically by detergents, high temperature, and some hydrophobic peptides (Coux et al., 1996
). To determine if the 7-residue peptides corresponding to PAN’s C-terminus cause gate-opening by the same mechanism as PAN, we tested whether the peptide must also contain the HbYX motif. When the Hb or the Y residue was replaced by an alanine, the peptide was not able to stimulate gate opening, as was found with PAN (). Also, replacement of the C-terminal arginine with an alanine did not reduce its activity, as was found with the corresponding mutation in PAN (). Furthermore, the 7-residue peptide from PAN’s C-terminus could not stimulate gate opening in proteasomes expressing the K66A mutation in the α-subunits (). Thus, gate opening by the C-terminal peptides and PAN shows the same requirements for an HbYX motif and a lysine 66 in the intersubunit pockets, indicating that they function by very similar mechanism.
Peptides from PAN’s C-terminus inhibit PAN-20S association
If these peptides induce gate opening by docking into the same sites in the α-ring as PAN’s C-termini, then these peptides should act as competitive inhibitors of the PAN-20S association. We therefore pre-incubated these peptides (250SM) with 20S proteasomes and R430W PAN (350nM) for five minutes prior to the addition of ATPγS, and then monitored complex formation by measuring polarization of PAN’s terminal tryptophan. The wild-type peptide inhibited complex formation nearly completely throughout the course of the experiment, while the inactive peptide containing an alanine in place of the conserved tyrosine did not inhibit ().
Since an additional alanine on PAN’s C-terminus prevents complex formation (Table S2
), it seemed likely that a free carboxyl group in this position is important for binding to the 20S. To test this possibility, we blocked the C-terminal carboxyl group on HLDVLYR by esterification with methanolic acid. After carboxy-esterification, the peptide completely lost its ability to inhibit polarization (). Thus a carboxyl group is essential for docking in the α-ring. Together, these observations establish that gate opening induced by peptides from PAN’s C-terminus is a specific response that requires a length of 7 residues, the conserved HbYX motif, a free carboxyl group and lysine 66 in the 20S’s α-ring. In related studies, we have used cryo-electron microscopy to confirm that the gate-opening peptides bind to the intersubunit pockets in the 20S’s α-ring adjacent to lys66 (Rabl et al, Ms in Preparation).
C-termini of PA26 bind but do not cause gate opening
Because the C-termini of PAN and PA26 both require lysine 66 in the α-subunits to associate with the 20S and activate gate opening, they are likely to bind to or act through the same sites (Forster et al., 2005
), even though PA28/26 lack the HbYX motif. Therefore, we studied under these same conditions the effects on gating of the seven residues peptide corresponding to the C-terminus of PA26, GTPHMVS, or eight residues peptides from the C-terminus of PA28α (which by itself forms a heptameric complex that induces gate opening in eukaryotic 20S proteasomes (Cascio et al., 2002
)). However, in contrast to the 7- or 8-residue peptides from PAN’s C-terminus, these peptides could not by themselves induce gate-opening at 250 μM () or even at 1mM (not shown). The failure of this PA26 peptide to stimulate gate opening is consistent with the prior findings that the C-terminal residues of PA26 are required for its association with the 20S, but that this binding does not induce gate opening, which requires the distant activation domain of PA26 (Zhang et al., 1998
To determine whether the heptapeptide from PA26 actually binds to the 20S, we measured fluorescence polarization of PAN R430W to test whether this C-terminal peptide could competitively inhibit PAN’s association with the 20S. After preincubation with the 20S, this peptide, like those from PAN’s C-terminus, was found to inhibit the polarization that was associated with PAN-20S complex formation (). However, this inhibition of polarization, unlike that by the C-terminal peptides from PAN, was temporary and lasted for only several minutes, presumably because the PA26-derived peptide had much lower affinity for the 20S than PAN and the PAN-derived C-terminal peptide.
C-terminal peptides from PAN and certain 19S ATPases induce gate opening in the mammalian 20S
Since the HbYX motif is also found in the C-termini of three of the ATPases in the 26S proteasome, it seemed likely that these C-termini regulate gating in the eukaryotic 20S in a similar fashion as PAN. We therefore tested if similar mechanisms function in the 26S particle, and if short peptides from PAN’s C-terminus could induce gate opening in proteasomes from rabbit muscle. In these particles, gate opening was monitored with the tripeptide substrate GGL-amc, which is excluded by the closed gate conformation. The 7 and 8 residue peptides from PAN were indeed able to cause a 5–6 fold increase in peptide entry, but smaller ones had no effect. Thus, the mammalian 20S showed the same length requirement for gate opening as did the archaeal 20S ().
To determine whether the ATPases in the 26S proteasome function similarly to PAN, we synthesized eight-residue peptides corresponding to the C-terminus of each of the mammalian 19S ATPases, Rpt1–6. Because such C-terminal peptides may also enter the 20S particle and compete with the fluorogenic substrates for binding to the active sites, we monitored the induction of gate opening with substrates specific for its different catalytic sites (i.e. chymotrypsin-like site: GGL-amc, caspase-like site: nLPnLD-amc, and trypsin-like site: LRR-amc) to reduce the chances that substrate competition would mask the effects on gate opening. The C-terminal peptides from Rpt2 and Rpt5, which contains the HbYX motif, strongly stimulated gate opening in the rabbit 20S proteasome, and did so to a greater extent than the C-terminal peptide from PAN (). Prior genetic studies had also implicated Rpt2 in regulation of gate-opening (Kohler et al, 2002). As expected, Rpt 4 and Rpt6 which do not contain the HbYX motif, could not induce gate opening. Rpt1 was found to induce gate opening weakly but only under some conditions, possibly because the residue preceding its penultimate tyrosine is a threonine (not a hydrophobic residue) and thus does not conform to the HbYX motif. Surprisingly, Rpt3, whose last three residues (…FYK) does contain the HbYX motif was never found to induce gate opening in mammalian 20S particles. Related observations indicated that lysines in the C-terminal (X) position do not support gate opening in mammalian 20S in contrast to the HbYX motif in PAN which functions with basic (but not acidic) residues in this position (DS and AG, data not shown). Thus, gate opening by the HbYX motif appears to function in 26S proteasomes, although gating seems to be regulated directly by only C-termini of Rpt2 and Rpt5, although other C-termini may contribute indirectly (e.g. by stabilizing the 26S complex) (see below).
The mammalian 19S ATPases’ C-termini appear to be essential for gate opening
To determine if the C-termini of the 19S ATPases, Rpt1–6, are also required for gate opening in mammalian 26S, we studied the effects of carboxypeptidase treatment of purified mammalian 19S/PA700 complex (kindly provided by George DeMartino) on its ability to induce gate opening in purified rabbit 20S proteasomes, in an experiment similar to that done with PAN (). The C-terminal residues of the ATPases Rpt3 and Rpt6 are lysines which should be removed by carboxypeptidase B, while those on Rpt2 (Leu), Rpt4 (Val), and possibly Rpt5 (Ala) should be removed by carboxypeptidase A. Treatment of the 19S complex with either carboxypeptidase A or B prior to addition of the 20S and ATPγS significantly reduced the ability of the 19S to stimulate peptide hydrolysis in the presence of ATPγS (). Thus, both classes of 19S C-termini seem important for the stimulation of gate opening. (In these experiments, carboxypeptidases B seemed more effective than A, but these effects cannot be compared quantitatively since the extent of removal of the different C-terminal residues by the two enzyme preparations may well differ.)
Figure 4 Carboxypeptidase induced truncation and mutations within the HbYX motif result in 26S instability and gating defects. A) Carboxypeptidase treatment of free mammalian 19S/PA700 inhibits its ability to stimulate peptide hydrolysis by the mammalian 20S in (more ...)
By contrast, treatment of the isolated 26S complex with carboxypeptidase A or B did not reduce the stimulation of peptide hydrolysis by ATPγS (not shown), presumably because the critical C-terminal residue in the 19S ATPases are also buried upon complex formation, as was found with the PAN-20S complex. These latter observations also indicate that the carboxypeptidase treatments did not destroy the integrity of the 19S complex or act indirectly by affecting subunits outside the 19S–20S interacting surfaces.
The C-terminal YX residues in the yeast 26S ATPases are required for gate opening
These findings suggest that the C-termini of multiple ATPase subunits are important in 26S formation and gating. To further test whether the terminal YX residues are necessary for 26S stability or gate-opening, we examined the effects of mutagenesis of the YX residues in the C-termini of the four ATPase subunits in S. cerevisiae
proteasomes. We substituted an alanine for the conserved penultimate tyrosine in the four ATPase subunits that contain this residue, Rpt1, Rpt2, Rpt3, and Rpt5, and purified the 26S proteasomes from these mutant strains using affinity tags (Leggett et al., 2002
). It is noteworthy that each of all these YA replacements in the four ATPase C-termini caused highly complex and distinct complex phenotypes related to protein degradation, but all allowed sufficient proteasome function for growth of the yeast (S.P. and D.F., unpublished data). Analysis of these phenotypic defects is beyond the scope of this study and will be published elsewhere. The activity of each mutant proteasome was assessed in nondenaturing gels. After electrophoresis, the gels were incubated in the presence of the fluorogenic substrate, suc-LLVY-amc, to assess the extent of complexation and gate opening (). Replacement of the conserved tyrosine in Rpt1 by an alanine appeared to cause a major defect in the stability of the 26S complex, since we were not able to isolate these particles by standard affinity purification methods. By contrast, the rpt2YA, rpt3YA
, and rpt5YA
mutant proteasomes were successfully purified by these approaches, but they all exhibited reduced suc-LLVY-amc hydrolysis. These defects in peptide hydrolysis reflect primarily decreased substrate entry and not simply reduced 26S proteasome levels, because equivalent proteasome loading was evident when the gels were stained, and the 26S proteasomes did not disassemble during electrophoresis or during incubation with substrate (see supplement and Fig. S2
). In addition, to test the capacity for gate opening, this assay was repeated in the presence of 0.02% SDS, which artificially induces gate opening (). The rpt3YA
mutant proteasomes showed a significant increase in peptide hydrolysis with SDS, but the rpt5YA
mutant showed only a limited response. Thus, the conserved tyrosines in each of the four ATPases appear important for regulating gate opening in the 20S particle, but their specific roles appear to differ.
A similar approach was then used to delete the C-terminal residues on the six 19S ATPases. Each of these mutants also had phenotypic defects, related to proteolysis, which will be analyzed elsewhere. The C-terminal deletion in Rpt3 resulted in a clear gating defect similar to that seen with the Tyr to Ala substitutions in Rpt2, Rpt3 and Rpt5 (). When the C-termini of the other ATPases Rpt4, Rpt5, and Rpt6, were truncated by a single C-terminal residue, there appeared to be stability problems in the 26S complex that reduced recovery (S.P. and D.F. unpublished data). Thus, the terminal YX residues are necessary for proper gating in the yeast 26S proteasome, and some appear necessary for its stability. While the terminal HbYX motifs in the 19S ATPases appear to play important functions in gating and complex formation that are consistent with the findings on PAN, the specific roles of the individual ATPases C-termini clearly differ from one another.