Kaye Truscott

Mitochondrial matrix proteostasis is linked to hereditary paraganglioma: LON‐mediated turnover of the human flavinylation factor SDH5 is regulated by its interaction with SDHA

Publisher: Wiley

Date: 10-01-2014

DOI: 10.1096/FJ.13-242420

Abstract: Mutations in succinate dehydrogenase (SDH) subunits and assembly factors cause a range of clinical conditions. One such condition, hereditary paraganglioma 2 (PGL2), is caused by a G78R mutation in the assembly factor SDH5. Although SDH5(G78R) is deficient in its ability to promote SDHA flavinylation, it has remained unclear whether impairment to its import, structure, or stability contributes to its loss of function. Using import-chase analysis in human mitochondria isolated from HeLa cells, we found that the import and maturation of human SDH5(G78R) was normal, while its stability was reduced significantly, with ~25% of the protein remaining after 180 min compared to ~85% for the wild-type protein. Notably, the metabolic stability of SDH5(G78R) was restored to wild-type levels by depleting mitochondrial LON (LONM). Degradation of SDH5(G78R) by LONM was confirmed in vitro however, in contrast to the in organello analysis, wild-type SDH5 was also rapidly degraded by LONM. SDH5 instability was confirmed in SDHA-depleted mitochondria. Blue native PAGE showed that imported SDH5(G78R) formed a transient complex with SDHA however, this complex was stabilized in LONM depleted mitochondria. These data demonstrate that SDH5 is protected from LONM-mediated degradation in mitochondria by its stable interaction with SDHA, a state that is dysregulated in PGL2.

Mitochondria Use Different Mechanisms for Transport of Multispanning Membrane Proteins through the Intermembrane Space

Publisher: Informa UK Limited

Date: 11-2003

DOI: 10.1128/MCB.23.21.7818-7828.2003

Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP.

Publisher: Springer Science and Business Media LLC

Date: 06-11-2020

DOI: 10.1038/S42003-020-01358-6

Abstract: Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been h ered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/β linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like β-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

Unfolded protein responses in bacteria and mitochondria: A central role for the ClpXP machine

Publisher: Wiley

Date: 26-10-2011

DOI: 10.1002/IUB.526

Abstract: In the crowded environment of a cell, the protein quality control machinery, such as molecular chaperones and proteases, maintains a population of folded and hence functional proteins. The accumulation of unfolded proteins in a cell is particularly harmful as it not only reduces the concentration of active proteins but also overburdens the protein quality control machinery, which in turn, can lead to a significant increase in nonproductive folding and protein aggregation. To circumvent this problem, cells use heat shock and unfolded protein stress response pathways, which essentially sense the change to protein homeostasis upregulating protein quality control factors that act to restore the balance. Interestingly, several stress response pathways are proteolytically controlled. In this review, we provide a brief summary of targeted protein degradation by AAA+ proteases and focus on the role of ClpXP proteases, particularly in the signaling pathway of the Escherichia coli extracellular stress response and the mitochondrial unfolded protein response.

Dual Function of Sdh3 in the Respiratory Chain and TIM22 Protein Translocase of the Mitochondrial Inner Membrane

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1016/J.MOLCEL.2011.09.025

Abstract: The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.

Insight into the RssB-Mediated Recognition and Delivery of σs to the AAA+ Protease, ClpXP

Publisher: MDPI AG

Date: 16-04-2020

DOI: 10.3390/BIOM10040615

Abstract: In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)—which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σS recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (βα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a “closed” state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.

Novel modification by L/F-tRNA-protein transferase (LFTR) generates a Leu/N-degron ligand in Escherichia coli

Publisher: Cold Spring Harbor Laboratory

Date: 27-07-2021

DOI: 10.1101/2021.07.26.453911

Abstract: The N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherchia coli the principal N-degron pathway is known as the Leu/N-degron pathway of which an Nt Leu is a key feature of the degron. Although the physiological role of the Leu/N-degron pathway is currently unclear, many of the components of the pathway are well defined. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (N d1 ) residue (Leu, Phe, Trp or Tyr) and an unstructured region which generally contains a hydrophobic element. Most N-degrons are generated from a pro-N-degron, either by endoproteolytic cleavage, or by enzymatic attachment of a N d1 residue (Leu or Phe) to the N-terminus of a protein (or protein fragment) by the enzyme Leu/Phe tRNA protein transferase (LFTR) in a non-ribosomal manner. Regardless of the mode of generation, all Leu/N-degrons are recognized by ClpS and delivered to the ClpAP protease for degradation. To date, only two physiological Leu/N-degron bearing substrates have been verified, one of which (PATase) is modified by LFTR. In this study, we have examined the substrate proteome of LFTR during stationary phase. From this analysis, we have identified several additional physiological Leu/N-degron ligands, including AldB, which is modified by a previously undescribed activity of LFTR. Importantly, the novel specificity of LFTR was confirmed in vitro , using a range of model proteins. Our data shows that processing of the Nt-Met of AldB generates a novel substrate for LFTR. Importantly, the LFTR-dependent modification of T 2 -AldB is essential for its turnover by ClpAPS, in vitro . To further examine the acceptor specificity of LFTR, we performed a systematic analysis using a series of peptide arrays. These data reveal that the identity of the second residue modulates substrate conjugation with positively charged residues being favored and negatively charged and aromatic residues being disfavored. Collectively, these findings extend our understanding of LFTR specificity and the Leu/N-degron pathway in E. coli .

Modification of PATase by L/F-transferase generates a ClpS-dependent N-end rule substrate in Escherichia coli

Publisher: Wiley

Date: 14-05-2009

DOI: 10.1038/EMBOJ.2009.134

The N-end rule adaptor protein ClpS from Plasmodium falciparum exhibits broad substrate specificity

Publisher: Wiley

Date: 23-09-2016

DOI: 10.1002/1873-3468.12382

Abstract: The N-end rule is a conserved protein degradation pathway that relates the metabolic stability of a protein to the identity of its N-terminal residue. Proteins bearing a destabilising N-terminal residue (N-degron) are recognised by specialised components of the pathway (N-recognins) and degraded by cellular proteases. In bacteria, the N-recognin ClpS is responsible for the specific recognition of proteins bearing an N-terminal destabilising residue such as leucine, phenylalanine, tyrosine or tryptophan. In this study, we show that the putative apicoplast N-recognin from Plasmodium falciparum (PfClpS), in contrast to its bacterial homologues, exhibits an expanded substrate specificity that includes recognition of the branched chain amino acid isoleucine.

Structural basis of N‐end rule substrate recognition in Escherichia coli by the ClpAP adaptor protein ClpS

Publisher: EMBO

Date: 17-04-2009

DOI: 10.1038/EMBOR.2009.62

Molecular and structural insights into an asymmetric proteolytic complex (ClpP1P2) from Mycobacterium smegmatis

Publisher: Springer Science and Business Media LLC

Date: 02-12-2019

DOI: 10.1038/S41598-019-53736-8

Abstract: The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis ( Msm ) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of Msm ClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.

LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

Publisher: Springer Science and Business Media LLC

Date: 02-12-2015

DOI: 10.1038/SREP17397

Abstract: Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPR mt ). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPR mt . Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPR mt , this pathway may preferentially act to promote chaperone mediated refolding of proteins.

A J-protein is an essential subunit of the presequence translocase–associated protein import motor of mitochondria

Publisher: Rockefeller University Press

Date: 24-11-2003

DOI: 10.1083/JCB.200308004

Abstract: Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase–associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70) the peripheral membrane protein Tim44 and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

Crystal structure of bacterial succinate:quinone oxidoreductase flavoprotein SdhA in complex with its assembly factor SdhE

Publisher: Proceedings of the National Academy of Sciences

Date: 07-03-2018

DOI: 10.1073/PNAS.1800195115

Abstract: Assembly factors play key roles in the biogenesis of many multisubunit protein complexes regulating their stability, activity, or incorporation of essential cofactors. The bacterial assembly factor SdhE (also known as Sdh5 or SDHAF2 in mitochondria) promotes covalent attachment of flavin adenine dinucleotide (FAD) to SdhA and hence the assembly of functional succinate:quinone oxidoreductase (also known as complex II). Here, we present the crystal structure of Escherichia coli SdhE bound to its client protein SdhA. This structure provides unique insight into SdhA assembly, whereby SdhE constrains unassembled SdhA in an “open” conformation, promoting covalent attachment of FAD, but renders the holoprotein incapable of substrate catalysis. These data also provide a structural explanation for the loss-of-function mutation, Gly78Arg, in SDHAF2, which causes hereditary paraganglioma 2.

The N-end rule pathway: From recognition by N-recognins, to destruction by AAA+proteases

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.BBAMCR.2011.07.002

Abstract: Intracellular proteolysis is a tightly regulated process responsible for the targeted removal of unwanted or damaged proteins. The non-lysosomal removal of these proteins is performed by processive enzymes, which belong to the AAA+superfamily, such as the 26S proteasome and Clp proteases. One important protein degradation pathway, that is common to both prokaryotes and eukaryotes, is the N-end rule. In this pathway, proteins bearing a destabilizing amino acid residue at their N-terminus are degraded either by the ClpAP protease in bacteria, such as Escherichia coli or by the ubiquitin proteasome system in the eukaryotic cytoplasm. A suite of enzymes and other molecular components are also required for the successful generation, recognition and delivery of N-end rule substrates to their cognate proteases. In this review we examine the similarities and differences in the N-end rule pathway of bacterial and eukaryotic systems, focusing on the molecular determinants of this pathway.

Diverse functions of mitochondrial AAA+ proteins: protein activation, disaggregation, and degradationThis paper is one of a selection of papers published in this special issue entitled 8th Internation

Publisher: Canadian Science Publishing

Date: 02-2010

DOI: 10.1139/O09-167

Abstract: In eukaryotes, mitochondria are required for the proper function of the cell and as such the maintenance of proteins within this organelle is crucial. One class of proteins, collectively known as the AAA+ (ATPases associated with various cellular activities) superfamily, make a number of important contributions to mitochondrial protein homeostasis. In this organelle, they contribute to the maturation and activation of proteins, general protein quality control, respiratory chain complex assembly, and mitochondrial DNA maintenance and integrity. To achieve such erse functions this group of ATP-dependent unfoldases utilize the energy from ATP hydrolysis to modulate the structure of proteins via unique domains and (or) associated functional components. In this review, we describe the current status of knowledge regarding the known mitochondrial AAA+ proteins and their role in this organelle.

PDIP38 is a novel adaptor-like modulator of the mitochondrial AAA+ protease CLPXP

Publisher: Cold Spring Harbor Laboratory

Date: 22-05-2020

DOI: 10.1101/2020.05.19.105320

Abstract: Polymerase δ interacting protein of 38 kDa (PDIP38) was originally identified in a yeast two hybrid screen as an interacting protein of DNA polymerase delta, more than a decade ago. Since this time several subcellular locations have been reported and hence its function remains controversial. Our current understanding of PDIP38 function has also been h ered by a lack of detailed biochemical or structural analysis of this protein. Here we show, that human PDIP38 is directed to the mitochondrion, where it resides in the matrix compartment, together with its partner protein CLPX. PDIP38 is a bifunctional protein, composed of two conserved domains separated by an α-helical hinge region (or middle domain). The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain (ZBD) of CLPX. In contrast, the C-terminal (DUF525) domain forms an Immunoglobin-like β-sandwich fold, which contains a highly conserved hydrophobic groove. Based on the physicochemical properties of this groove, we propose that PDIP38 is required for the recognition (and delivery to CLPXP) of proteins bearing specific hydrophobic degrons, potentially located at the termini of the target protein. Significantly, interaction with PDIP38 stabilizes the steady state levels of CLPX in vivo . Consistent with these data, PDIP38 inhibits the LONM-mediated turnover of CLPX in vitro. Collectively, our findings shed new light on the mechanistic and functional significance of PDIP38, indicating that in contrast to its initial identification as a nuclear protein, PIDP38 is a bona fide mitochondrial adaptor protein for the CLPXP protease.

Perrault syndrome type 3 caused by diverse molecular defects in CLPP

Publisher: Springer Science and Business Media LLC

Date: 27-08-2018

DOI: 10.1038/S41598-018-30311-1

Abstract: The maintenance of mitochondrial protein homeostasis (proteostasis) is crucial for correct cellular function. Recently, several mutations in the mitochondrial protease CLPP have been identified in patients with Perrault syndrome 3 (PRLTS3). These mutations can be arranged into two groups, those that cluster near the docking site (hydrophobic pocket, Hp) for the cognate unfoldase CLPX (i.e. T145P and C147S) and those that are adjacent to the active site of the peptidase (i.e. Y229D). Here we report the biochemical consequence of mutations in both regions. The Y229D mutant not only inhibited CLPP-peptidase activity, but unexpectedly also prevented CLPX-docking, thereby blocking the turnover of both peptide and protein substrates. In contrast, Hp mutations cause a range of biochemical defects in CLPP, from no observable change to CLPP activity for the C147S mutant, to dramatic disruption of most activities for the “gain-of-function” mutant T145P - including loss of oligomeric assembly and enhanced peptidase activity.

Pupylation of PafA or Pup inhibits components of the Pup‐Proteasome System

Publisher: Wiley

Date: 17-12-2017

DOI: 10.1002/1873-3468.12930

Abstract: The pupylation of cellular proteins plays a crucial role in the degradation cascade via the Pup‐Proteasome system ( PPS ). It is essential for the survival of Mycobacterium smegmatis under nutrient starvation and, as such, the activity of many components of the pathway is tightly regulated. Here, we show that Pup, like ubiquitin, can form polyPup chains primarily through K61 and that this form of Pup inhibits the ATP ase‐mediated turnover of pupylated substrates by the 20S proteasome. Similarly, the autopupylation of PafA (the sole Pup ligase found in mycobacteria) inhibits its own enzyme activity hence, pupylation of PafA may act as a negative feedback mechanism to prevent substrate pupylation under specific cellular conditions.

Control of Protein Function through Regulated Protein Degradation: Biotechnological and Biomedical Applications

Publisher: S. Karger AG

Date: 2013

DOI: 10.1159/000352043

Abstract: Targeted protein degradation is crucial for the correct function and maintenance of a cell. In bacteria, this process is largely performed by a handful of ATP-dependent machines, which generally consist of two components - an unfoldase and a peptidase. In some cases, however, substrate recognition by the protease may be regulated by specialized delivery factors (known as adaptor proteins). Our detailed understanding of how these machines are regulated to prevent uncontrolled degradation within a cell has permitted the identification of novel antimicrobials that dysregulate these machines, as well as the development of tunable degradation systems that have applications in biotechnology. Here, we focus on the physiological role of the ClpP peptidase in bacteria, its role as a novel antibiotic target and the use of protein degradation as a biotechnological approach to artificially control the expression levels of a protein of interest.

Mitochondrial Presequence Translocase: Switching between TOM Tethering and Motor Recruitment Involves Tim21 and Tim17

Publisher: Elsevier BV

Date: 03-2005

DOI: 10.1016/J.CELL.2005.01.011

Abstract: The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.

The bacterial N-end rule pathway: expect the unexpected

Publisher: Wiley

Date: 30-03-2010

DOI: 10.1111/J.1365-2958.2010.07120.X

Abstract: The N-end rule pathway is a highly conserved process that operates in many different organisms. It relates the metabolic stability of a protein to its N-terminal amino acid. Consequently, amino acids are described as either 'stabilizing' or 'destabilizing'. Destabilizing residues are organized into three hierarchical levels: primary, secondary, and in eukaryotes - tertiary. Secondary and tertiary destabilizing residues act as signals for the post-translational modification of the target protein, ultimately resulting in the attachment of a primary destabilizing residue to the N-terminus of the protein. Regardless of their origin, proteins containing N-terminal primary destabilizing residues are recognized by a key component of the pathway. In prokaryotes, the recognition component is a specialized adaptor protein, known as ClpS, which delivers target proteins directly to the ClpAP protease for degradation. In contrast, eukaryotes use a family of E3 ligases, known as UBRs, to recognize and ubiquitylate their substrates resulting in their turnover by the 26S proteasome. While the physiological role of the N-end rule pathway is largely understood in eukaryotes, progress on the bacterial pathway has been slow. However, new interest in this area of research has invigorated several recent advances, unlocking some of the secrets of this unique proteolytic pathway in prokaryotes.

Pam16 has an essential role in the mitochondrial protein import motor

Publisher: Springer Science and Business Media LLC

Date: 15-02-2004

DOI: 10.1038/NSMB735

Insertion and Assembly of Human Tom7 into the Preprotein Translocase Complex of the Outer Mitochondrial Membrane

Publisher: Elsevier BV

Date: 11-2002

DOI: 10.1074/JBC.M205613200

Anti-adaptors use distinct modes of binding to inhibit the RssB-dependent turnover of RpoS (σS) by ClpXP

Publisher: Frontiers Media SA

Date: 23-04-2015

DOI: 10.3389/FMOLB.2015.00015

Conserved residues in the N‐domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding

Publisher: Wiley

Date: 14-02-2008

DOI: 10.1111/J.1742-4658.2008.06304.X

Abstract: Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N‐domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N‐domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N‐domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide‐binding groove within the N‐domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS‐mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N‐domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.

Substrate recognition and processing by a Walker B mutant of the human mitochondrial AAA+ protein CLPX

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1016/J.JSB.2012.06.001

Abstract: The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, including CLPXP, some of which contribute to protein maturation and quality control. Currently however, the substrates and the physiological roles of mitochondrial CLPXP in humans, has remained elusive. Similarly, the mechanism by which these ATP-dependent proteases recognize their substrates currently remains unclear. Here we report the characterization of a Walker B mutation in human CLPX, in which the highly conserved glutamate was replaced with alanine. This mutant protein exhibits improved interaction with the model unfolded substrate casein and several putative physiological substrates in vitro. Although this mutant lacks ATPase activity, it retains the ability to mediate casein degradation by hCLPP, in a fashion similar to the small molecule ClpP-activator, ADEP. Our functional dissection of hCLPX structure, also identified that most model substrates are recognized by the N-terminal domain, although some substrates bypass this step and dock, directly to the pore-1 motif. Collectively these data reveal, that despite the difference between bacterial and human CLPXP complexes, human CLPXP exhibits a similar mode of substrate recognition and is deregulated by ADEPs.

Discovery Projects - Grant ID: DP0450051

Start Date: 05-2004

End Date: 09-2009

Amount: $1,100,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0775758

Start Date: 2007

End Date: 12-2007

Amount: $587,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0770013

Start Date: 2007

End Date: 12-2010

Amount: $263,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110103936

Start Date: 2011

End Date: 12-2016

Amount: $425,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150104639

Start Date: 02-2015

End Date: 12-2019

Amount: $320,600.00

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT0992033

Start Date: 07-2009

End Date: 12-2014

Amount: $686,400.00

Funder: Australian Research Council