BBA - Molecular Cell Research (v.1823, #1)

Structure and function of the AAA+ nucleotide binding pocket by Petra Wendler; Susanne Ciniawsky; Malte Kock; Sebastian Kube (2-14).
Members of the diverse superfamily of AAA+ proteins are molecular machines responsible for a wide range of essential cellular processes. In this review we summarise structural and functional data surrounding the nucleotide binding pocket of these versatile complexes. Protein Data Bank (PDB) structures of closely related AAA+ ATPase are overlaid and biologically relevant motifs are displayed. Interactions between protomers are illustrated on the basis of oligomeric structures of each AAA+ subgroup. The possible role of conserved motifs in the nucleotide binding pocket is assessed with regard to ATP binding and hydrolysis, oligomerisation and inter-subunit communication. Our comparison indicates that in particular the roles of the arginine finger and sensor 2 residues differ subtly between AAA+ subgroups, potentially providing a means for functional diversification. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► We provide an overview of the current classification of AAA+ proteins. ► The characteristic features of the AAA+ nucleotide binding pocket are presented. ► We assemble biochemical data summarising the effects of site directed mutations in the nucleotide binding pocket. ► We assess the structural conservation of AAA+ domains and their nucleotide binding pockets. ► PDB structures of oligomeric AAA+ proteins are examined regarding protomer interactions at the nucleotide binding pocket.
Keywords: AAA+ ATPases; Nucleotide binding pocket; Structural analysis; Functional analysis;

ClpXP, an ATP-powered unfolding and protein-degradation machine by Tania A. Baker; Robert T. Sauer (15-28).
ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies. This article is part of a Special Issue entitled: AAA ATPases: Structure and function.► Ring hexamers of ClpX unfold and then translocate proteins into ClpP for degradation. ► A narrow pore restricts ClpP activity unless ClpX/ClpA or acyldepsipeptides bind. ► ClpX recognizes peptide tags or degrons in protein substrates and adaptor proteins. ► Asymmetric ATP hydrolysis by one ClpX subunit powers mechanical protein unfolding. ► Crystallography and single-molecule results reveal detailed aspects of mechanism.
Keywords: Targeted degradation; Energy-dependent proteolysis; Protein unfolding; Protein translocation;

The elusive middle domain of Hsp104 and ClpB: Location and function by Morgan E. DeSantis; James Shorter (29-39).
Hsp104 in yeast and ClpB in bacteria are homologous, hexameric AAA+ proteins and Hsp100 chaperones, which function in the stress response as ring-translocases that drive protein disaggregation and reactivation. Both Hsp104 and ClpB contain a distinctive coiled-coil middle domain (MD) inserted in the first AAA+ domain, which distinguishes them from other AAA+ proteins and Hsp100 family members. Here, we focus on recent developments concerning the location and function of the MD in these hexameric molecular machines, which remains an outstanding question. While the atomic structure of the hexameric assembly of Hsp104 and ClpB remains uncertain, recent advances have illuminated that the MD is critical for the intrinsic disaggregase activity of the hexamer and mediates key functional interactions with the Hsp70 chaperone system (Hsp70 and Hsp40) that empower protein disaggregation. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Discussion and review of hexameric models of Hsp104 and ClpB. ► Discussion of function of middle domain of Hsp104 and ClpB in Hsp70 collaboration. ► Discussion of Hsp70-independent functions of middle domain of Hsp104 and ClpB.
Keywords: Hsp104; ClpB; Hexamer; Aggregate; Prion;

Structure and function of the bacterial AAA protease FtsH by Sina Langklotz; Ulrich Baumann; Franz Narberhaus (40-48).
Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA+ proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► FtsH is a conserved AAA protease found in eubacteria, mitochondria and chloroplasts. ► As typical for AAA+ proteases, bacterial FtsH forms barrel-shaped homo-hexamers. ► FtsH is membrane-anchored and crucial for membrane protein quality control. ► In E. coli, FtsH is essential due to regulation of lipopolysaccharide biosynthesis. ► FtsH plays a role in regulation of the E. coli heat shock response.
Keywords: FtsH; Proteolysis; AAA protein; LpxC; LPS biosynthesis; Heat shock;

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. This article is part of a Special Issue entitled: AAA ATPases: Structure and function.► AAA proteases form hexameric, membrane-embedded complexes built up of homologous subunits. ► AAA proteases have dual activities acting as quality control and processing enzymes. ► AAA proteases recognize the folding state of substrate proteins. ► Intersubunit communication coordinates ATP hydrolysis within AAA ring and ensures maximal force generation.
Keywords: AAA+ protein; ATP-dependent protease; Mitochondria; Structure; Quality control of protein;

Multitasking in the mitochondrion by the ATP-dependent Lon protease by Sundararajan Venkatesh; Jae Lee; Kamalendra Singh; Irene Lee; Carolyn K. Suzuki (56-66).
The AAA+ Lon protease is a soluble single-ringed homo-oligomer, which represents the most streamlined operational unit mediating ATP-dependent proteolysis. Despite its simplicity, the architecture of Lon proteases exhibits a species-specific diversity. Homology modeling provides insights into the structural features that distinguish bacterial and human Lon proteases as hexameric complexes from yeast Lon, which is uniquely heptameric. The best-understood functions of mitochondrial Lon are linked to maintaining proteostasis under normal metabolic conditions, and preventing proteotoxicity during environmental and cellular stress. An intriguing property of human Lon is its specific binding to G-quadruplex DNA, and its association with the mitochondrial genome in cultured cells. A fraction of Lon preferentially binds to the control region of mitochondrial DNA where transcription and replication are initiated. Here, we present an overview of the diverse functions of mitochondrial Lon, as well as speculative perspectives on its role in protein and mtDNA quality control. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Homology modeling of human Lon predicts a hexameric complex. ► Role of Lon as a protein quality control protease. ► Lon-mediated proteolysis in the regulation of cellular metabolism. ► Potential role of Lon in mitochondrial DNA maintenance and expression. ► Lon expression changes in human disease.
Keywords: AAA+; Mitochondria; ATP-dependent protease; Lon; Unfolded protein response (UPR); mtDNA;

Proteasomal AAA-ATPases: Structure and function by Shoshana Bar-Nun; Michael H. Glickman (67-82).
The 26S proteasome is a chambered protease in which the majority of selective cellular protein degradation takes place. Throughout evolution, access of protein substrates to chambered proteases is restricted and depends on AAA-ATPases. Mechanical force generated through cycles of ATP binding and hydrolysis is used to unfold substrates, open the gated proteolytic chamber and translocate the substrate into the active proteases within the cavity. Six distinct AAA-ATPases (Rpt1–6) at the ring base of the 19S regulatory particle of the proteasome are responsible for these three functions while interacting with the 20S catalytic chamber. Although high resolution structures of the eukaryotic 26S proteasome are not yet available, exciting recent studies shed light on the assembly of the hetero-hexameric Rpt ring and its consequent spatial arrangement, on the role of Rpt C-termini in opening the 20S ‘gate’, and on the contribution of each individual Rpt subunit to various cellular processes. These studies are illuminated by paradigms generated through studying PAN, the simpler homo-hexameric AAA-ATPase of the archaeal proteasome. The similarities between PAN and Rpts highlight the evolutionary conserved role of AAA-ATPase in protein degradation, whereas unique properties of divergent Rpts reflect the increased complexity and tighter regulation attributed to the eukaryotic proteasome. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► AAA-ATPases, generators of mechanical force, are key players in protein degradation throughout evolution. ► Archaeal PAN homo-hexameric ring provides insights into structure and function of eukaryotic hetero-hexameric Rpt ring. ► Exciting findings show the role of the C-termini of proteasomal AAA-ATPases in opening the proteolytic chamber ‘gate’. ► Novel discoveries on assembly pathways lead to revised spatial arrangement of eukaryotic hetero-hexameric Rpt ring. ► Functional studies emphasize the unique contribution of each individual Rpt subunit to various cellular processes.
Keywords: AAA-ATPase; Proteasome; 19S regulatory particle; PAN;

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. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► N-end rule substrates are recognized by specific binding proteins known as N-recognins. ► N-recognins contain specialized binding sites for the recognition of Type 1 and Type 2 N-degrons. ► N-recognins deliver these substrates to ClpAP in bacterial or the 26S proteasome in eukaryotes, for degradation.
Keywords: AAA + protein superfamily; N-end rule pathway; Protein degradation; ClpS; UBR box; Substrate binding;

The biogenesis of ribosomes is a fundamental cellular process, which provides the molecular machines that synthesize all cellular proteins. The assembly of eukaryotic ribosomes is a highly complex multi-step process that requires more than 200 ribosome biogenesis factors, which mediate a broad spectrum of maturation reactions. The participation of many energy-consuming enzymes (e.g. AAA-type ATPases, RNA helicases, and GTPases) in this process indicates that the expenditure of energy is required to drive ribosome assembly. While the precise function of many of these enzymes remains elusive, recent progress has revealed that the three AAA-type ATPases involved in 60S subunit biogenesis are specifically dedicated to the release and recycling of distinct biogenesis factors. In this review, we will highlight how the molecular power of yeast Drg1, Rix7, and Rea1 is harnessed to promote the release of their substrate proteins from evolving pre-60S particles and, where appropriate, discuss possible catalytic mechanisms. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Structural and functional properties of AAA-ATPases in ribosome biogenesis are summarized. ► The AAA-ATPases Rea1, Rix7 and Drg1 are essential for ribosome biogenesis in yeast. ► Rix7 and Drg1 are related to Cdc48, while Rea1 shares similarity to motor protein dynein. ► Rea1, Rix7 and Drg1 promote the release of biogenesis factors from nucleolar, nucleoplasmic and cytoplasmic pre-60S intermediates. ► The release of maturation factors by AAA-ATPases is critical for downstream maturation of pre-60S particles.
Keywords: Ribosome assembly; AAA-ATPase; Drg1/Afg2; Rix7/NVL; Rea1/Mdn1/Midasin;

The R2TP complex: Discovery and functions by Yoshito Kakihara; Walid A. Houry (101-107).
The two closely related AAA + family ATPases Rvb1 and Rvb2 are part of several critical multiprotein complexes, and, thus, are involved in a wide range of cellular processes including chromatin remodelling, telomerase assembly, and snoRNP biogenesis. It was found that Rvb1 and Rvb2 form a tight functional complex with Pih1 (Protein interacting with Hsp90) and Tah1 (TPR-containing protein associated with Hsp90), which are two Hsp90 interactors. We named the complex R2TP. The complex was originally isolated from Saccharomyces cerevisiae and was, subsequently, identified in mammalian cells. R2TP was found to be required for box C/D snoRNP biogenesis in yeast and mammalian cells. More recently, several studies revealed that the complex is also involved in multiple biological processes including apoptosis, phosphatidylinositol-3 kinase-related protein kinase (PIKK) signalling, and RNA polymerase II assembly. In this review, we describe the discovery of the complex and discuss the emerging critical roles that R2TP plays in distinct cellular processes. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► The R2TP (Rvb1-Rvb2-Tah1-Pih1) complex was identified in 2005 as an Hsp90 associated complex. ► The R2TP complex is conserved from yeast to higher eukaryotes. ► The complex is involved in box C/D snoRNP biogenesis. ► The complex is required for phosphatidylinositol-3 kinase-related protein kinase (PIKK) signalling. ► R2TP is involved in RNA polymerase II assembly.
Keywords: Rvb1; Rvb2; Tah1; Pih1; R2TP; Complex assembly;

Coupling AAA protein function to regulated gene expression by Nicolas Joly; Nan Zhang; Martin Buck; Xiaodong Zhang (108-116).
AAA proteins (ATPases Associated with various cellular Activities) are involved in almost all essential cellular processes ranging from DNA replication, transcription regulation to protein degradation. One class of AAA proteins has evolved to adapt to the specific task of coupling ATPase activity to activating transcription. These upstream promoter DNA bound AAA activator proteins contact their target substrate, the σ54-RNA polymerase holoenzyme, through DNA looping, reminiscent of the eukaryotic enhance binding proteins. These specialised macromolecular machines remodel their substrates through ATP hydrolysis that ultimately leads to transcriptional activation. We will discuss how AAA proteins are specialised for this specific task. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Discuss the ATPase cycle and how ATPase activities are relayed to substrate binding. ► Discuss specific residues and motifs that play distinct roles in ATP binding, hydrolysis and regulation. ► Discuss how ATPase activity is utilised in remodelling the substrate. ► Discuss how the AAA + activities are regulated.
Keywords: AAA ATPase; Structure–function relationship; RNA polymerase; Transcriptional activator; Enhancer binding protein; Sigma54;

The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in all eukaryotic cells that transforms ATP hydrolysis into mechanic power to unfold and pull proteins against physical forces, which make up a protein's structure and hold it in place. From the many cellular processes, Cdc48 is involved in, its function in endoplasmic reticulum associated protein degradation (ERAD) is understood best. This quality control process for proteins of the secretory pathway scans protein folding and discovers misfolded proteins in the endoplasmic reticulum (ER), the organelle, destined for folding of these proteins and their further delivery to their site of action. Misfolded lumenal and membrane proteins of the ER are detected by chaperones and lectins and retro-translocated out of the ER for degradation. Here the Cdc48 machinery, recruited to the ER membrane, takes over. After polyubiquitylation of the protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1–Npl4 pulls the misfolded protein out and away from the ER membrane and delivers it to down-stream components for degradation by a cytosolic proteinase machine, the proteasome. The known details of the Cdc48–Ufd1–Npl4 motor complex triggered process are subject of this review article. This article is part of a Special Issue entitled: AAA ATPases: Structure and function.►Cdc48 is a type II AAA-type ATPase with two ATP binding domains. ►From the many cellular functions of Cdc48 its involvement in ERAD is understood best. ►Cdc48 function is driven by several substrate recruiting and substrate processing co-factors. ►Cdc48 is a molecular motor with ratcheting and segregase activity.
Keywords: AAA-type ATPase; Cdc48; p97/VCP; Cdc48–Ufd1–Npl4 complex; ER-associated protein degradation (ERAD); Ubiquitin–proteasome-system (UPS);

Regulation of p97 in the ubiquitin–proteasome system by the UBX protein-family by Patrik Kloppsteck; Caroline A. Ewens; Andreas Förster; Xiaodong Zhang; Paul S. Freemont (125-129).
The AAA protein p97 is a central component in the ubiquitin–proteasome system, in which it is thought to act as a molecular chaperone, guiding protein substrates to the 26S proteasome for degradation. This function is dependent on association with cofactors that are specific to the different biological pathways p97 participates in. The UBX-protein family (ubiquitin regulatory X) constitutes the largest known group of p97 cofactors. We propose that the regulation of p97 by UBX-proteins utilizes conserved structural features of this family. Firstly, they act as scaffolding subunits in p97-containing multiprotein complexes, by providing additional interaction motifs. Secondly, they provide regulation of multiprotein complex assembly and we suggest two possible models for p97 substrate recruitment in the UPS pathway. Lastly, they impose constraints on p97 and its interaction with substrates and further cofactors. These features allow the regulation, within the UPS, of the competitive interactions on p97, a regulation that is crucial to allow the diverse functionality of p97. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► p97's key role in the UPS is facilitated by association with adaptor proteins. ► p97 availability is highly regulated to facilitate its many independent functions. ► Formation of p97 complexes is sequential and highly regulated. ► UBX proteins have a direct effect on p97 behavior.
Keywords: p97/VCP; Ubiquitin–proteasome system; UBX;

Recent advances in p97/VCP/Cdc48 cellular functions by Kunitoshi Yamanaka; Yohei Sasagawa; Teru Ogura (130-137).
p97/VCP/Cdc48 is one of the best-characterized type II AAA (ATPases associated with diverse cellular activities) ATPases. p97 is suggested to be a ubiquitin-selective chaperone and its key function is to disassemble protein complexes. p97 is involved in a wide variety of cellular activities. Recently, novel functions, namely autophagy and mitochondrial quality control, for p97 have been uncovered. p97 was identified as a causative factor for inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) and more recently as a causative factor for amyotrophic lateral sclerosis (ALS). In this review, we will summarize and discuss recent progress and topics in p97 functions and the relationship to its associated diseases. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► p97 as a ubiquitin-selective chaperone in the ubiquitin-proteasome system. ► Role of p97/CDC-48 in the sex determination pathway in C. elegans. ► Extraction of Aurora B kinase from chromatin by p97. ► p97/VCP as a causative factor for IBMPFD and ALS.
Keywords: p97/VCP/Cdc48; Autophagy; Mitochondria; IBMPFD; ALS; TDP-43;

The AAA-ATPase Cdc48/p97 controls a large array of cellular functions including protein degradation, cell division, membrane fusion through its ability to interact with and control the fate of ubiquitylated proteins. More recently, Cdc48/p97 also appeared to be involved in autophagy, a catabolic cell response that has long been viewed as completely distinct from the Ubiquitine/Proteasome System. In particular, conjugation by ubiquitin or ubiquitin-like proteins as well as ubiquitin binding proteins such as Cdc48/p97 and its cofactors can target degradation by both catabolic pathways. This review will focus on the recently described functions of Cdc48/p97 in autophagosome biogenesis as well as selective autophagy. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► The Cdc48 chaperone plays a major role in the ubiquitin/protesome pathway. ► Recently, Cdc48/p97 also appeared to be involved in autophagy, another catabolic pathway. ► This review will focus on the functions of Cdc48 in autophagosome biogenesis and selective autophagy.
Keywords: Cdc48; Autophagy; Ubiquitin; Proteasome;

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p–Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p. This article is part of a Special Issue entitled: AAA ATPases: structure and function.
Keywords: AAA peroxin; CHO cell mutant; Peroxisome biogenesis; Peroxisome biogenesis disorder; PTS1-receptor shuttling; Ubiquitination;

The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAA-type ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Pex1p and Pex6p are type-II AAA-proteins with a conserved second AAA-domain. ► Pex1p and Pex6p are crucial for peroxisomal matrix protein import. ► Pex1p and Pex6p form a heteromeric complex. ► The yeast Pex1p-Pex6p-complex is recruited to peroxisomes by Pex15p. ►The Pex1p-Pex6p-complex acts as dislocase for the peroxisomal import receptor Pex5p.
Keywords: AAA-type ATPases; PEX; Peroxin; Peroxisome biogenesis; Protein translocation; Ubiquitination;

Requirements for the catalytic cycle of the N-ethylmaleimide-Sensitive Factor (NSF) by Chunxia Zhao; Everett C. Smith; Sidney W. Whiteheart (159-171).
The N-ethylmaleimide-Sensitive Factor (NSF) was one of the initial members of the ATPases Associated with various cellular Activities Plus (AAA+) family. In this review, we discuss what is known about the mechanism of NSF action and how that relates to the mechanisms of other AAA+ proteins. Like other family members, NSF binds to a protein complex (i.e., SNAP–SNARE complex) and utilizes ATP hydrolysis to affect the conformations of that complex. SNAP–SNARE complex disassembly is essential for SNARE recycling and sustained membrane trafficking. NSF is a homo-hexamer; each protomer is composed of an N-terminal domain, NSF-N, and two adjacent AAA-domains, NSF-D1 and NSF-D2. Mutagenesis analysis has established specific roles for many of the structural elements of NSF-D1, the catalytic ATPase domain, and NSF-N, the SNAP–SNARE binding domain. Hydrodynamic analysis of NSF, labeled with (Ni2+-NTA)2-Cy3, detected conformational differences in NSF, in which the ATP-bound conformation appears more compact than the ADP-bound form. This indicates that NSF undergoes significant conformational changes as it progresses through its ATP-hydrolysis cycle. Incorporating these data, we propose a sequential mechanism by which NSF uses NSF-N and NSF-D1 to disassemble SNAP–SNARE complexes. We also illustrate how analytical centrifugation might be used to study other AAA+ proteins. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Compilation of mutagenesis data in different domains of NSF. ► New conformational change data for NSF during ATP hydrolysis. ► New concept for understanding NSF function.
Keywords: NSF; Membrane trafficking; ATPase; SNAP; SNARE;

Structure and function of the membrane deformation AAA ATPase Vps4 by Christopher P. Hill; Markus Babst (172-181).
The ATPase Vps4 belongs to the type-I AAA family of proteins. Vps4 functions together with a group of proteins referred to as ESCRTs in membrane deformation and fission events. These cellular functions include vesicle formation at the endosome, cytokinesis and viral budding. The highly dynamic quaternary structure of Vps4 and its interactions with a network of regulators and co-factors has made the analysis of this ATPase challenging. Nevertheless, recent advances in the understanding of the cell biology of Vps4 together with structural information and in vitro studies are guiding mechanistic models of this ATPase. This article is part of a Special Issue entitled:AAA ATPases: structure and function.► Vps4 is a type-I AAA ATPases that functions with ESCRT-III in the deformation and fission of membranes. ► Vps4-mediated membrane fission plays an important role for MVB vesicle formation, cytokinesis and viral budding. ► Vps4 posses a dynamic quaternary structure that is regulated by the bound nucleotide and co-factors.
Keywords: ESCRT; MVB pathway; Cytokinesis; Viral budding; Protein trafficking; Endosome;

Dynein is a large cytoskeletal motor protein that belongs to the AAA + (ATPases associated with diverse cellular activities) superfamily. While dynein has had a rich history of cellular research, its molecular mechanism of motility remains poorly understood. Here we describe recent X-ray crystallographic studies that reveal the architecture of dynein's catalytic ring, mechanical linker element, and microtubule binding domain. This structural information has given rise to new hypotheses on how the dynein motor domain might change its conformation in order to produce motility along microtubules. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► An overview and comparison of recent dynein crystal structures. ► Proposal of allosteric communication mechanisms of dynein based on other AAA proteins. ► Discussion of models of processive dynein motility on microtubules based on structural evidence.
Keywords: Dynein; Microtubule; Molecular motor; AAA+ ATPase; Protein structure;

The AAA ATPase spastin links microtubule severing to membrane modelling by Jennifer H. Lumb; James W. Connell; Rachel Allison; Evan Reid (192-197).
In 1999, mutations in the gene encoding the microtubule severing AAA ATPase spastin were identified as a major cause of a genetic neurodegenerative condition termed hereditary spastic paraplegia (HSP). This finding stimulated intense study of the spastin protein and over the last decade, a combination of cell biological, in vivo, in vitro and structural studies have provided important mechanistic insights into the cellular functions of the protein, as well as elucidating cell biological pathways that might be involved in axonal maintenance and degeneration. Roles for spastin have emerged in shaping the endoplasmic reticulum and the abscission stage of cytokinesis, in which spastin appears to couple membrane modelling to microtubule regulation by severing. This article is part of a Special Issue entitled: AAA ATPases: structure and function.► Spastin is a microtubule severing ATPase and mutations in the gene encoding spastin cause axonal degeneration. ► N‐terminal spastin domains serve to recruit it to cellular sites of action. ► A hydrophobic domain localizes spastin to the endoplasmic reticulum. ► The MIT domain recruits spastin to endosomes and the cytokinetic midbody. ► Spastin regulates microtubules in relation to sites of membrane modelling.
Keywords: Cytokinesis; Endoplasmic reticulum morphogenesis; Axonopathy; ESCRT complex; Hairpin hydrophobic domain; Hereditary spastic paraplegia;