Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes

Striebel, Frank; Kress, Wolfgang; Weber‐Ban, Eilika

doi:10.1016/j.sbi.2009.02.006

Cited by 124 publications

(106 citation statements)

References 57 publications

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“…Similar results were obtained for self-complemented FimA. 4 Hence, FimAta is one of the most stable proteins known. Thus, maybe not too surprisingly, degradation was neither detectable in presence of the ClpA Walker B variants nor in the presence of ClpAwt.…”

Section: Fimata-li-ssra)supporting

confidence: 79%

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Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Kress

Mutschler²,

Weber‐Ban

2009

Journal of Biological Chemistry

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ClpA is a ring-shaped hexameric chaperone that binds to both ends of the protease ClpP and catalyzes the ATP-dependent unfolding and translocation of substrate proteins through its central pore into the ClpP cylinder. Here we study the relevance of ATP hydrolysis in the two ATPase domains of ClpA. We designed ClpA Walker B variants lacking ATPase activity in the first (D1) or the second ATPase domain (D2) without impairing ATP binding. We found that the two ATPase domains of ClpA operate independently even in the presence of the protease ClpP or the adaptor protein ClpS. Notably, ATP hydrolysis in the first ATPase module is sufficient to process a small, single domain protein of low stability. Substrate proteins of moderate local stability were efficiently processed when D1 was inactivated. However, ATP hydrolysis in both domains was required for efficiently processing substrates of high local stability. Furthermore, we provide evidence for the ClpS-dependent directional translocation of N-end rule substrates from the N to C terminus and propose a mechanistic model for substrate handover from the adaptor protein to the chaperone.The chaperone ClpA is a member of the AAA ϩ protein family (ATPase-associated with various cellular activities) and catalyzes the energy-dependent degradation of proteins through interaction with the protease ClpP in Escherichia coli (1-3). Like many other AAA proteins (4), ClpA oligomerizes into a ring structure, shaping a central pore through which substrate proteins are routed into the proteolytic core ClpP. This process involves unfolding and translocation of the substrate protein and requires the consumption of ATP. AAA proteins can be grouped into class I (two ATPase domains) and class II (one ATPase domain) ATPases. The fact that some ATPases have only one AAA module whereas others seem to require two ATPase domains stimulated researchers to investigate the roles and interdependence of the two ATP-binding modules in class I AAA proteins like Hsp104 and ClpB (5-8). ClpA also features two ATPase domains, termed D1 and D2. They are highly homologous, but differences in the amino acid sequence around the conserved regions in D1 and D2 suggested that they might have a somewhat different function (9). As was shown for several other class I members, ClpA assembles into its oligomeric state only upon binding of nucleotide (10). Indeed, substituting the invariant lysine in the Walker A motif demonstrated that nucleotide binding to D1 triggers ClpA hexamerization, whereas ATP turnover is mainly catalyzed by D2 (11, 12). However, mutations in the Walker A motif also abolish or drastically decrease the affinity for ATP, making it impossible to distinguish between effects due to ATP binding and those due to ATP hydrolysis.To study the role of ATP hydrolysis in both ATPase domains uncoupled from nucleotide binding events, we designed ClpA Walker B variants that lack the ability to hydrolyze ATP in either D1 (ClpAE286A), D2 (ClpAE565A), or both domains (ClpAE286A/E565A) but still bind ATP in bot...

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Section: Fimata-li-ssra)supporting

confidence: 79%

“…Like many other AAA proteins (4), ClpA oligomerizes into a ring structure, shaping a central pore through which substrate proteins are routed into the proteolytic core ClpP. This process involves unfolding and translocation of the substrate protein and requires the consumption of ATP.…”

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confidence: 99%

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Kress

Mutschler²,

Weber‐Ban

2009

Journal of Biological Chemistry

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“…The Clp machinery has two oligomeric components: A barrel-shaped tetradecameric protease core with the catalytic sites sequestered inside the complex and hexameric ring-like ATP-dependent chaperones. The chaperones recognize specific substrates with or without the aid of adaptors, unfold these substrates, and translocate them into the proteolytic core for degradation (Baker and Sauer, 2006;Striebel et al, 2009). Compartmentalization of the proteolytic sites within the core complex, and coupling with chaperones and associated factors for substrate delivery, enable targeted protein degradation within the cell.…”

Section: Introductionmentioning

confidence: 99%

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

et al. 2011

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The caseinolytic protease (Clp) protease system has been expanded in plant plastids compared with its prokaryotic progenitors. The plastid Clp core protease consists of five different proteolytic ClpP proteins and four different noncatalytic ClpR proteins, with each present in one or more copies and organized in two heptameric rings. We determined the exact subunit composition and stoichiometry for the intact core and each ring. The chloroplast ClpP/R protease was affinity purified from clpr4 and clpp3 Arabidopsis thaliana null mutants complemented with C-terminal StrepII-tagged versions of CLPR4 and CLPP3, respectively. The subunit stoichiometry was determined by mass spectrometry-based absolute quantification using stable isotope-labeled proteotypic peptides generated from a synthetic gene. One heptameric ring contained ClpP3,4,5,6 in a 1:2:3:1 ratio. The other ring contained ClpP1 and ClpR1,2,3,4 in a 3:1:1:1:1 ratio, resulting in only three catalytic sites. These ClpP1/R1-4 proteins are most closely related to the two subunits of the cyanobacterial P3/R complex and the identical P:R ratio suggests conserved adaptation. Furthermore, the plant-specific C-terminal extensions of the ClpP/R subunits were not proteolytically removed upon assembly, suggesting a regulatory role in Clp chaperone interaction. These results will now allow testing ClpP/R structure-function relationships using rationale design. The quantification workflow we have designed is applicable to other protein complexes.

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“…The eukaryotic proteasome is essential and represents the main processive protease in the cytosol, while in bacteria multiple architecturally related proteases coexist [3]. The construction and functional principle is shared: a proteolytic cylinder enclosing the protease active sites associates at both ends with a regulatory ATPase particle [4,5].…”

Section: Introductionmentioning

confidence: 99%

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

et al. 2009

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Edited by Miguel De la RosaKeywords: Prokaryotic ubiquitin-like protein Mpa ARC Proteasome NMR Mycobacterium tuberculosis a b s t r a c tThe mycobacterial ubiquitin-like protein Pup is coupled to proteins, thereby rendering them as substrates for proteasome-mediated degradation. The Pup-tagged proteins are recruited by the proteasomal ATPase Mpa (also called ARC). Using a combination of biochemical and NMR methods, we characterize the structural determinants of Pup and its interaction with Mpa, demonstrating that Pup adopts a range of extended conformations with a short helical stretch in its C-terminal portion. We show that the N-terminal coiled-coil domain of Mpa makes extensive contacts along the central region of Pup leaving its N-terminus unconstrained and available for other functional interactions. Structured summary:MINT-7262427: pup (uniprotkb:B6DAC1) binds (MI:0407) to mpa (uniprotkb:Q0G9Y7) by pull down (MI:0096) MINT-7262440: mpa (uniprotkb:Q0G9Y7) and pup (uniprotkb:B6DAC1) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

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Controlled destruction: AAA+ ATPases in protein degradation from bacteria to eukaryotes

Cited by 124 publications

References 57 publications

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Both ATPase Domains of ClpA Are Critical for Processing of Stable Protein Structures

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

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