Conserved Pore Residues in the AAA Protease FtsH Are Important for Proteolysis and Its Coupling to ATP Hydrolysis

Yamada-Inagawa, Tomoko; Okuno, Takashi; Karata, Kiyonobu; Yamanaka, Kunitoshi; Ogura, Teru

doi:10.1074/jbc.m308327200

Cited by 124 publications

(127 citation statements)

References 51 publications

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“…The mechanical force for this process is generated from repeated cycles of ATP binding and hydrolysis. Substrates are pulled through the narrow channel of the unfoldase via attachment to pore loops located at the entrance and in the interior of the channel (65,(67)(68)(69)(70), which also play a role in substrate recognition in some AAA+ machines (5,(71)(72)(73). In the case of VAT, mutations of pore loop residues Y264 in D1 and W541/V542 in D2 cause severe defects on substrate unfolding and translocation (18).…”

Section: Discussionmentioning

confidence: 99%

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Huang

Ripstein

Augustyniak

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.T he AAA+ enzymes (ATPases associated with a variety of cellular activities) use the energy of ATP hydrolysis to carry out a myriad of different biological functions that are critical to cellular homeostasis. These molecular machines are typically barrel-shaped, containing a narrow channel that serves to traffic substrates through the enzyme (1-4). In the case of the heat shock protein 104 (Hsp104) AAA+ class of chaperone, for example, substrates are dissociated from aggregates via a pulling motion that forces individual polypeptide chains through the central pore of the enzyme (1, 5). The unfolded proteins that emerge subsequently refold spontaneously or are acted on by additional chaperones that aid in their refolding (6). Other AAA+ enzymes act synergistically with proteases, unfolding targeted substrates and passing them directly to the proteases, where they are subsequently degraded (7). In this way, proteins that have become damaged or that are no longer required can be removed before their accumulation disrupts cellular function (8). A variety of different AAA+ unfoldases have been characterized, including Rpt1-6 in the 19S proteasome regulatory particle in eukaryotes (9-11), PAN in archaea (12-14), Mpa/ARC in Actinobacteria (15), ClpA/X (2) and HslU (3) in bacteria, and VAT in the Thermoplasma acidophilum archaebacterium (16)(17)(18)(19)(20).VAT is an ∼500-kDa homohexamer, with each monomer containing two tandem AAA+ domains, referred to as D1 and D2, and an N-terminal domain (NTD) distinct from the NTD of other AAA+ enzymes (16) (Fig. 1A). Both D1 and D2 are homologous to the single AAA+ modules of PAN and Rpt1-6 (1, 16). Biochemical studies have shown that VAT unfolds globular proteins and interacts directly with the 20S core particle (CP) of the proteasome, leading to enhanced proteolytic activity for both folded and ...

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Section: Discussionmentioning

confidence: 99%

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Huang

Ripstein

Augustyniak

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Position 234 carries a conserved aromatic residue in all FtsH orthologs from bacteria, chloroplasts, and mitochondria. In E. coli, mutation of the equivalent amino acid (Phe-225) to alanine gives a protein that is not any more able to degrade 32 but can still hydrolyze unfolded substrates like resorufin-casein, whereas substitution of this phenylalanine by charged residues (e.g., Glu) abolishes activity completely (27).…”

Section: Substrate Access Is Granted By a Narrow Cleft Guarded By Aromentioning

confidence: 99%

The molecular architecture of the metalloprotease FtsH

Bieniossek

Schalch

Bumann

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

151

136

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The ATP-dependent integral membrane protease FtsH is universally conserved in bacteria. Orthologs exist in chloroplasts and mitochondria, where in humans the loss of a close FtsH-homolog causes a form of spastic paraplegia. FtsH plays a crucial role in quality control by degrading unneeded or damaged membrane proteins, but it also targets soluble signaling factors like 32 and -CII. We report here the crystal structure of a soluble FtsH construct that is functional in caseinolytic and ATPase assays. The molecular architecture of this hexameric molecule consists of two rings where the protease domains possess an all-helical fold and form a flat hexagon that is covered by a toroid built by the AAA domains. The active site of the protease classifies FtsH as an Asp-zincin, contrary to a previous report. The different symmetries of protease and AAA rings suggest a possible translocation mechanism of the target polypeptide chain into the interior of the molecule where the proteolytic sites are located.AAA ͉ protease ͉ protein degradation ͉ x-ray A TP-dependent proteases play crucial roles in protein quality control and regulation (for reviews, see refs. 1 and 2). One of these is FtsH, initially described as a temperature-sensitive and cell-division-defective mutant, which is also called HflB, named after a high-frequency of lysogenization locus of bacteriophage . FtsH is an integral membrane protease found in bacteria, chloroplasts, and mitochondria (reviewed in ref.3) (Fig. 6, which is published as supporting information on the PNAS web site). In bacteria, FtsH malfunction causes severe phenotypes like cell division defects and growth arrest (4, 5) (reviewed in ref.3). Deletion of the human mitochondrial homolog paraplegin, which shares 40% sequence identity with FtsH from Escherichia coli, is responsible for an autosomal recessive form of hereditary spastic paraplegia (6). The N terminus of FtsH contains two transmembrane helices followed by an AAA module (ATPases associated with various cellular activities), including the SRH (second region of homology) (3). The C-terminal part of the polypeptide chain bears the HEXXH motif that is characteristic for Zn-dependent metalloproteases, where the two histidines coordinate to the zinc ion and the glutamate serves as a catalytic base. In bacteria, the AAA and protease domains are located on the cytosolic side of the membrane. Of the five ATP-dependent proteases in E. coli, HslVU, Lon, ClpXP, ClpAP, and FtsH, the last is the only one that is essential and universally conserved in bacteria. It degrades membrane proteins like the uncomplexed SecY subunit of translocase (7), the a-subunit of F o F 1 -ATPase (8), and the photosystem in chloroplasts (9), therefore playing an important role in the quality control of membrane proteins. Further targets comprise regulatory soluble proteins such as 32 (10, 11) or -CII transcriptional activator protein (12). All these substrates are degraded in an ATP-dependent manner where the energy is used for pulling the proteins out of the membran...

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“…The targeting signals are recognized by the proteases through loops in the central channel of the ATPase ring [55][56][57][58]. The best characterized of these is a loop containing the sequence GYVG, which is highly conserved in most proteolytic AAA+ ATPases and has been implicated directly in protein unfolding and translocation [55].…”

Section: Targeting Signals In the Primary Sequence Of The Substratesmentioning

confidence: 99%

Protein targeting to ATP-dependent proteases

Inobe

Matouschek

2008

Current Opinion in Structural Biology

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ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be useful to separate two steps: substrate binding to the protease and initiation of degradation.To be active, newly synthesized protein chains must fold into three-dimensional structures, but regulated unfolding is also critically important in some biological processes, such as protein degradation by ATP-dependent proteases and protein translocation across membranes [1]. Unfolding is required during degradation because the proteolytic sites of the ATP-dependent proteases are sequestered deep inside the proteases' structures and accessible only through narrow openings. Similarly, unfolding is required during several translocation processes because the protein import channels in some organelles are not wide enough for native proteins to fit through them. The mechanisms of unfolding in both types of processes are similar to each other but different from that of unfolding induced by heat or chemical denaturants. Here we discuss how the requirement for protein unfolding during degradation affects the way ATPdependent proteases select their substrates. ATP-dependent proteasesATP-dependent proteases degrade short-lived regulatory proteins and thereby control cellular processes such as signal transduction, cell cycle, and gene transcription. The proteases also clear misfolded and aggregated proteins from the cell and produce some of the peptides to be displayed at cell surface as part of adaptive immune response. In eukaryotes, these functions are performed mainly by the proteasome. In prokaryotes and the organelles of eukaryotes, the functions are fulfill by analogues of the proteasome, such as the ClpAP, ClpXP, HslUV, FtsH, and Lon proteases. Although ATP-dependent proteases show only relatively little sequence identity, they share a common architecture [2].The ATP-dependent proteases all form large multisubunit particles (Figure 1). In the simplest case, FtsH protease, the particle consists 6 copies of a 71 kDa subunit forming a complex of approximately 425 kDa, and in the most complex case, the proteasome, the particle consists of some 40 different subunits forming a complex of 2 MDa molecular weight [3,4]. The subunits are mostly arranged in six or seven subunit rings that stack on top of each other to

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Conserved Pore Residues in the AAA Protease FtsH Are Important for Proteolysis and Its Coupling to ATP Hydrolysis

Cited by 124 publications

References 51 publications

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

The molecular architecture of the metalloprotease FtsH

Protein targeting to ATP-dependent proteases

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