Coordinated ATP hydrolysis within m-AAA protease complexes
To characterize the regulation of ATP hydrolysis by m
-AAA proteases, we introduced point mutations into the Walker A motifs of the conserved AAA domains of Yta10 and Yta12 (Yta10K334A
). While Δyta10
cells expressing Yta10 and Yta12 were able to grow under respiratory conditions, coexpression of Yta10K334A
did not restore respiratory growth ((Tatsuta et al., 2007
); ). In contrast, expression of either the Yta10 or the Yta12 mutant variant in combination with the wild type form of the respective other subunit promoted respiratory growth of Δyta10
cells, suggesting that the activity of the m
-AAA protease is maintained if only one type of subunit is active (). Mutations in Walker B motifs of Yta10 or Yta12 (Yta10E388Q
) had a strikingly different effect. While mutations in both subunits inhibited respiratory growth as was observed for the Walker A mutants (Tatsuta et al., 2007
), coexpression of either Walker B mutant variant with the wild type form of the respective other subunit did not or only weakly restore growth on non-fermentable carbon sources ().
Coordinated ATP hydrolysis within yeast m-AAA protease complexes
We purified m
-AAA protease complexes to define the effect of these mutations on the ATPase activity ((Tatsuta et al., 2007
); Fig. S1
). Regardless of the relative expression of Yta10 and Yta12, both subunits were present at equimolar concentrations in purified complexes (Arlt et al., 1996
) that form hexameric rings (Fig. S1
and unpublished observations) similar to most other AAA+ proteins (Hanson and Whiteheart, 2005
; Erzberger and Berger, 2006
). Genetic and biochemical evidence suggests an alternating rather than a stochastic subunit arrangement within m
-AAA protease complexes (Fig. S2
). Mutations in the Walker A or B motifs of Yta10 and Yta12 did not affect assembly of m
-AAA complexes but abolished ATP hydrolysis completely when present in both subunits (; Fig. S1
). Similar to other AAA+ proteins, replacement of the conserved lysine residue within the Walker A motif impaired ATP binding, whereas ATP is trapped if the conserved glutamate residue within the Walker B motif is mutated (; (Weibezahn et al., 2003
; Dalal et al., 2004
-AAA proteases with mutant Walker A motifs in one subunit exhibited ATPase activities of ~30% when compared to wild type, irrespective of which subunit carried the mutation (). Thus, both subunits can hydrolyze ATP independently. The reduced ATPase activity of the mutant m
-AAA proteases is still sufficient to maintain respiratory growth (). Mutations in the Walker B motif of Yta10 reduced the ATPase activity of m
-AAA complexes to ~30% (), whereas the corresponding mutation in Yta12 almost completely inhibited the ATPase activity of the mutant m
-AAA protease despite the presence of wild type Yta10 in these complexes (). Yta10 still binds ATP under these conditions as suggested by the almost unaltered Km
for ATP of these complexes (Fig. S3
). We therefore conclude that ATP binding to Yta12 blocks ATP hydrolysis by Yta10, pointing to a regulatory mechanism which coordinates ATP hydrolysis by m
-AAA protease subunits.
To investigate whether coordinated ATP hydrolysis requires two different AAA domains or can also occur in homo-oligomeric AAA rings, we replaced AAA domains of Yta10 or Yta12 by the AAA domains of the respective other subunit. Expression of the Yta12 hybrid subunit containing the AAA domain of Yta10 (termed H10; ) supported respiratory growth of Δyta10
cells only when coexpressed with Yta10 () indicating formation of functional complexes (Fig. S4
). Similar results were obtained when the AAA domain of Yta10 was replaced by that of Yta12 (; Fig. S4
). Thus, m
-AAA protease complexes containing six identical AAA domains are functionally active in vivo
. Mutations in the Walker A motifs of H10 or H12 affected respiratory growth only slightly (). Mutations in Walker B motifs of H10 and H12, however, strongly inhibited respiratory growth, ATPase and the proteolytic activities of the assembled proteases in vivo
( and Fig. S5
), suggesting that ATP binding impairs ATP hydrolysis in homo-oligomeric AAA rings.
ATP binding coordinates ATP hydrolysis in human m-AAA complexes
To examine whether coordinated ATP hydrolysis is conserved in human m
-AAA proteases, we expressed paraplegin and AFG3L2 or variants thereof harboring mutations in the AAA domain in Δyta10
cells. In this experimental setting, respiratory growth depends on paraplegin subunits within the hetero-oligomeric m
-AAA isoenzyme when inactive AFG3L2 variants are expressed (Koppen et al., 2007
). This allowed us to monitor effects of specific mutations within the AAA domain of AFG3L2 on paraplegin activity in vivo
. Coexpression of C-terminally hexahistadine tagged paraplegin with the Walker A AFG3L2 mutant partially restored respiratory growth of Δyta10
cells (). In contrast, respiratory growth was completely abolished when paraplegin was coexpressed with AFG3L2 harboring a mutation in the Walker B motif (). Mutations in Walker B but not Walker A motifs of AFG3L2 inhibited almost completely the ATPase activity of purified hetero-oligomeric m
-AAA complexes in vitro
(). Similar effects were observed when m
-AAA proteases with analogous mutant subunits of paraplegin were analyzed (). Thus, only mutations in the Walker B motif inhibited ATP hydrolysis in hetero-oligomeric human protease complexes, consistent with an inhibitory effect of ATP binding on adjacent subunits.
Dominant negative effects of mutations in the Walker B motif on homo- and hetero-oligomeric human m-AAA proteases
Next, we assessed whether homo-oligomeric AFG3L2 complexes are regulated similarly. Δyta10
cells expressing AFG3L2 variants concomitantly from two plasmids were able to grow on non-fermentable carbon sources, while mutations in Walker A- and B motifs introduced into both Afg3l2
genes abolished respiratory growth (). Interestingly, a Walker A variant of AFG3L2 (AFG3L2K354A
) maintained respiratory growth more efficiently than a Walker B variant (AFG3L2E408Q
) upon coexpression with AFG3L2 (). This dominant negative effect was substantiated by monitoring in vivo
processing of cytochrome c
peroxidase (Ccp1) and of the ribosomal protein MrpL32, two known substrates of the m
-AAA protease in yeast mitochondria ((Esser et al., 2002
; Nolden et al., 2005
To unambiguously demonstrate assembly of mutant and wild type AFG3L2 subunits, we coexpressed AFG3L2 chimera containing a C-terminal DHFR domain together with AFG3L2 variants which harbor a C-terminal hexahistidine tag and mutations in Walker A-or Walker B-motifs. The fusion of the DHFR domain to the C-terminus of AFG3L2 did not impair respiratory competence (), but allowed assessment of the relative amount of wild type subunits within assembled complexes. DHFR- and his-tagged AFG3L2 assembled stochastically regardless of the presence of mutations in his-tagged subunits (). We determined ATPase activities of purified AFG3L2 complexes and normalized them to the number of (DHFR-tagged) wild type subunits (). These experiments revealed that AFG3L2 subunits carrying a mutation in the Walker A motif did not affect the ATPase activity of wild type subunits in a complex (). Conversely, subunits harboring a mutation in their Walker B motif exert a dominant negative effect and inhibit ATP hydrolysis by homo-oligomeric AFG3L2 complexes ().
We therefore conclude that coordinated ATP hydrolysis by AAA domains of m-AAA protease subunits is conserved from yeast to man, and applies to both homo- and hetero-oligomeric m-AAA complexes. While individual m-AAA subunits can hydrolyze ATP independent of each other, ATP hydrolysis is inhibited when neighboring m-AAA subunits are in the ATP-bound state.
Defining intersubunit signaling within m-AAA ring complexes
Coordinated ATP hydrolysis within m-AAA rings requires sensing of nucleotide binding by neighboring subunits. To identify amino acid residues involved in intersubunit communication, an unbiased genetic screen was performed to identify suppressor mutations in Yta10, which would alleviate the ATPase activity block of Yta10 imposed upon ATP binding to Yta12 (). We reasoned that an Yta10 variant, which carries a mutation in an amino acid residue involved in sensing the nucleotide-bound state of Yta12, would be able to hydrolyze ATP and support respiratory growth, even if Yta12 harbors a mutation in the Walker B motif. Notably, mutations of conserved amino acid residues abolishing ATPase activity would not be isolated using such a gain-of-function screening procedure.
Identification of amino acid residues in Yta10 involved in intersubunit signaling
Nine individual mutations in Yta10 were identified, which suppress the inhibitory effect of the Walker B Yta12 mutant (). These suppressor mutations are located in three defined regions within the AAA domain of Yta10 (; Fig. S6
). Mutations replacing the conserved arginine finger residue (Yta10R447
) were isolated most frequently. Other mutations cluster to a region (residues 418–423) at the end of the helix α7 and preceding sensor-1 of Yta10. This region, which we termed the ISS (i
ignaling) motif, is conserved within the classical AAA clade of proteins (Fig. S6
). The third set of suppressor mutations affects a flexible loop after the Walker B motif of Yta10. This loop is highly charged, protrudes into the central pore of the AAA ring and corresponds structurally to the pore loop-2 present in many AAA+ ATPases ((Hinnerwisch et al., 2005
; Martin et al., 2008
); Fig. S6
). Consistent with a role for intersubunit signaling, suppressor mutations did not significantly affect ATP binding by purified m
-AAA proteases but did restore, at least partially, the intrinsic ATPase activities of complexes harboring Yta12E448Q
subunits upon assembly with Yta10 (; Fig. S7
To examine the spatial relationship of the affected amino acid residues, we generated an atomic model of the hetero-oligomeric yeast m
-AAA protease from the crystal structure of a soluble domain of FtsH (Bieniossek et al., 2006
; Suno et al., 2006
). In our model, the ISS motif in helix α7 is located close to the arginine finger R447 making possible an interaction of D421 with R450 (). Moreover, helix α7 directly follows the pore loop-2 and the Walker B motif. This arrangement suggests that a potential interaction between D421 and R450 couples movements of the arginine finger to amino acid residues in the ATP binding pocket of Yta10. This hypothesis was substantiated by mutations introduced at both sites. The growth defect caused by Yta12E448Q
was at least partially suppressed by replacing R450 with alanine or a deletion of five amino acids within the pore loop-2 ().
Taken together, our results are consistent with a signaling cascade from Yta12 to Yta10, which involves sensing of the nucleotide-bound state of Yta12 by the arginine finger R447 of Yta10, and transmission of this information through R450, D421 and helix α7 to the Walker B motif and the ATP binding pocket of Yta10.
Intersubunit signaling within Yta12
In order to identify amino acid residues in Yta12 required for intersubunit communication, we performed a similar genetic screen to identify intragenic suppressors for the growth defect associated with the Yta12 Walker B mutant (). Nine suppressor mutations in Yta12E448Q
were identified supporting respiratory growth (). A mutation of the conserved glycine residue within the Walker A motif of Yta12 (G391S) suppresses the inhibitory effect of the Walker B mutation most likely due to impaired nucleotide binding to Yta12 (). Replacing the originally introduced glutamine in the Walker B motif (E448Q) with arginine also allowed respiratory cell growth (). The majority of identified suppressor mutations, however, affect the region surrounding the conserved pore loop-1 of Yta12 (), which is crucial for substrate translocation through the central pore of many AAA+ ATPases (Song et al., 2000
; Yamada-Inagawa et al., 2003
; Weibezahn et al., 2004
; Hinnerwisch et al., 2005
; DeLaBarre et al., 2006
; Tatsuta et al., 2007
; Martin et al., 2008
Identification of amino acid residues in Yta12 involved in intersubunit signaling
The suppressor mutations E416K and R428K restored respiratory growth most efficiently and increased the ATPase activities of purified m
-AAA proteases at least to some extent (). Concurrently, they did not inhibit ATP binding to mutant m
-AAA complexes and, therefore, could be directly involved in intersubunit signaling (). In our atomic model of the m
-AAA protease, the region preceding the pore loop-1 is close to E448 in the Walker B motif suggesting that many suppressor mutations may affect coordination and positioning of the nucleotide within the ATP binding pocket of Yta12 (). Interestingly, the corresponding region was recently proposed to regulate the ATPase activity of related AAA+ proteins in response to ligand binding (Joly et al., 2008
; Zhang and Wigley, 2008
). Consistently, the identification of intragenic suppressors adjacent to the pore loop-1 of Yta12 points to an intimate coupling between coordinated ATP hydrolysis within the AAA+ ring and substrate handling.
Substrate-specific requirement for intersubunit coordination
To determine the role of coordinated ATP hydrolysis within the AAA+ ring for substrate handling directly, we analyzed the effect of an impaired intersubunit signaling for various m
-AAA protease activities, which differ in their energy requirements (; (Leonhard et al., 2000
; Tatsuta et al., 2007
)). For instance, processing of soluble MrpL32 requires only insertion of N-terminal segments of MrpL32 into the proteolytic chamber of the m
-AAA protease. It depends on ATP hydrolysis but can be mediated by m
-AAA variants with low ATPase activity (Nolden et al., 2005
). On the other hand, processing of Ccp1 or turnover of the misfolded inner membrane protein Yme2ΔC requires ATP-dependent membrane dislocation of substrates prior to proteolysis (Leonhard et al., 2000
; Tatsuta et al., 2007
Intersubunit signaling within Yta12 and substrate handling by the m-AAA protease
We expressed Yta12 variants harboring mutations in the Walker A- (K394A) or Walker B- (E448Q) motif, or a suppressor mutation preceding the pore loop-1 (E416K) in Δyta12 cells and monitored their activity upon assembly with Yta10. In agreement with its dominant negative effect on cell growth and ATPase activity of the m-AAA protease (), expression of Yta12E448Q abolished processing of both MrpL32 and Ccp1 as well as turnover of Yme2ΔC (). A mutation in the Walker A motif of Yta12 severely affected the ATPase activity of the m-AAA protease and inhibited Ccp1 processing and turnover of Yme2ΔC (). However, the mutant m-AAA complex allowed respiratory growth () and supported at least some processing of MrpL32 (), providing direct evidence for the ability of individual m-AAA subunits to cleave substrates. Strikingly, the suppressor mutation E416K preceding the pore loop-1 did not interfere with processing of MrpL32 but inhibited processing of Ccp1 and degradation of Yme2ΔC strongly (). This is not caused by a deficient ATPase activity of the m-AAA protease, since the ATPase activity was not affected by the Yta12E416K mutation ().
Next, we performed similar experiments with m-AAA protease variants harboring mutations in Yta10 (). Mutations in the Walker A- (K334A) and Walker B- (E388Q) motif of Yta10 reduced significantly the ATPase and proteolytic activities of mutant m-AAA protease complexes (). Ccp1 processing and Yme2ΔC turnover was almost completely inhibited, whereas Yta10K334A allowed MrpL32 processing to an extent sufficient to maintain respiratory growth (). Mutations in the pore loop-2 (R396G) or the ISS motif (D421V), which prevent intersubunit signaling and suppress the dominant negative effect of Yta12E448Q, affected the ATPase activity to a lesser extent than mutations in either Walker A- or Walker B-motifs (). Consistently, efficient maturation of MrpL32 occurred in these cells (). However, defective intersubunit signaling impaired severely Ccp1 processing and the degradation of Yme2ΔC ().
Intersubunit signaling in Yta10 and substrate handling by the m-AAA protease
Taken together, our experiments suggest that intersubunit signaling and a coordinated ATP hydrolysis within the AAA ring is dispensable for proteolytic activity per se, but essential for the dislocation of substrates by the m-AAA protease.
Coupling of ATPase and substrate processing by pore loop-1
To further dissect the coupling between ATP hydrolysis within the AAA ring and substrate handling, we focused on mutant m-AAA proteases carrying mutations in Walker B motifs of Yta10. These complexes exerted ATPase activity but were barely active in vivo (, ), suggesting an impaired coupling between ATPase and proteolytic activities. We screened for intragenic suppressor mutations in the Walker B mutant of Yta10, which restore the respiratory growth, using a similar strategy as described above. Seven independent suppressor mutations in Yta10E388Q were identified, which cause an amino acid exchange in the pore loop-1 region (). We purified m-AAA protease complexes composed of wild type Yta12 and mutant Yta10F361A E388Q subunits which harbored mutations in both the Walker B motif and the pore loop-1. Strikingly, ATPase activities of these complexes were slightly reduced when compared to complexes harboring Yta10E388Q subunits, but processing of MrpL32 in vivo was significantly increased (). These results suggest that ATP binding to Yta10 and pore loop-1 functions in substrate translocation is intimately coupled.
Role of pore loop-1 for coupling of ATPase and proteolytic activities of m-AAA proteases