Torsins: not your typical AAA+ ATPases

Abstract

Torsin ATPases (Torsins) belong to the widespread AAA+ (ATPases associated with a variety of cellular activities) family of ATPases, which share structural similarity but have diverse cellular functions. Torsins are outliers in this family because they lack many characteristics of typical AAA+ proteins, and they are the only members of the AAA+ family located in the endoplasmic reticulum and contiguous perinuclear space. While it is clear that Torsins have essential roles in many, if not all metazoans, their precise cellular functions remain elusive. Studying Torsins has significant medical relevance since mutations in Torsins or Torsin-associated proteins result in a variety of congenital human disorders, the most frequent of which is early-onset torsion (DYT1) dystonia, a severe movement disorder. A better understanding of the Torsin system is needed to define the molecular etiology of these diseases, potentially enabling corrective therapy. Here, we provide a comprehensive overview of the Torsin system in metazoans, discuss functional clues obtained from various model systems and organisms and provide a phylogenetic and structural analysis of Torsins and their regulatory cofactors in relation to disease-causative mutations. Moreover, we review recent data that have led to a dramatically improved understanding of these machines at a molecular level, providing a foundation for investigating the molecular defects underlying the associated movement disorders. Lastly, we discuss our ideas on how recent progress may be utilized to inform future studies aimed at determining the cellular role(s) of these atypical molecular machines and their implications for dystonia treatment options.     

AAA+ ATPases in the initiation of DNA replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

AAA+ ATPases in the Initiation of DNA Replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

AAA+ ATPases: structural insertions under the magnifying glass

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The Archaeal Proteasome Is Regulated by a Network of AAA ATPases

The proteasome is the central machinery for targeted protein degradation in archaea, Actinobacteria, and eukaryotes. In its basic form, it consists of a regulatory ATPase complex and a proteolytic core particle. The interaction between the two is governed by an HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) at the C terminus of the ATPase subunits, which stimulates gate opening of the proteasomal α-subunits. In archaea, the proteasome-interacting motif is not only found in canonical proteasome-activating nucleotidases of the PAN/ARC/Rpt group, which are absent in major archaeal lineages, but also in proteins of the CDC48/p97/VAT and AMA groups, suggesting a regulatory network of proteasomal ATPases. Indeed, Thermoplasma acidophilum, which lacks PAN, encodes one CDC48 protein that interacts with the 20S proteasome and activates the degradation of model substrates. In contrast, Methanosarcina mazei contains seven AAA proteins, five of which, both PAN proteins, two out of three CDC48 proteins, and the AMA protein, function as proteasomal gatekeepers. The prevalent presence of multiple, distinct proteasomal ATPases in archaea thus results in a network of regulatory ATPases that may widen the substrate spectrum of proteasomal protein degradation.     

Conserved arginine residues implicated in ATP hydrolysis, nucleotide-sensing, and inter-subunit interactions in AAA and AAA+ ATPases

Arginines are a recurrent feature of the active sites and subunit interfaces of the ATPase domains of AAA and AAA+ proteins. In particular family members these residues occupy two or more, of four key sites in the vicinity of the ATP cofactor, where they transduce the chemical events of ATP binding and hydrolysis into a mechanochemical outcome. Structural and biochemical analyses have led to the proposal of molecular mechanisms in which these conserved arginines play crucial roles. Comparative studies, however, point to functional divergence for each of these conserved arginines. In this review, we will discuss what is known about these critical arginines and what can be concluded about their role in the function of AAA and AAA+ proteins.     

AAA+ ATPases: achieving diversity of function with conserved machinery

AAA+ adenosine triphosphatases (ATPases) are molecular machines that perform a wide variety of cellular functions. For instance, they can act in vesicle transport, organelle assembly, membrane dynamics and protein unfolding. In most cases, the ATPase domains of these proteins assemble into active ring-shaped hexamers. As AAA+ proteins have a common structure, a central issue is determining how they use conserved mechanistic principles to accomplish specific biological actions. Here, we review the features and motifs that partially define AAA+ domains, describe the cellular activities mediated by selected AAA+ proteins and discuss the recent work, suggesting that various AAA+ machines with very different activities employ a common core mechanism. The importance of this mechanism to human health is demonstrated by the number of genetic diseases caused by mutant AAA+ proteins.     

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

Novel structural and functional insights into the MoxR family of AAA+ ATPases

The MoxR family of AAA+ ATPases is widespread among bacteria and archaea, although their cellular functions are not well characterized. Based on recent studies, MoxR ATPases are proposed to have chaperone-like function for the maturation of specific protein complexes or for the insertion of cofactors into proteins. MoxR proteins have been found to be important modulators of multiple stress response pathways in different organisms. For example, the respective MoxR proteins have been found to play important roles in the cell envelope stress response in Rhizobium leguminosarum, in the oxidative stress, acid stress, and heat stress responses in Francisella tularensis, in the acid stress and stringent responses in Escherichia coli, in viral tail formation in the crenarchaeal Acidianus two-tailed virus, and in the utilization of carbon monoxide as the sole carbon source by the Gram-negative chemolithoautotrophe Oligotropha carboxidovorans. Recent structural studies on the MoxR proteins from E. coli and Cytophaga hutchinsonii show the unique spatial arrangement of the αβα and all-α subdomains of the AAA+ domain in these proteins compared to the typical arrangement found in canonical AAA+ proteins such as HslU. The spatial organization of the subdomains in the AAA+ domain of MoxR proteins is similar to that found in the ATPase component of the magnesium chelatase complexes, possibly suggesting a similar mechanism of function. In this review, we provide an overview of the newly identified functions and the newly obtained structures of MoxR AAA+ ATPases.     

Distinct roles for the AAA ATPases NSF and p97 in the secretory pathway

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATP-hydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion.     

Assembly of the AAA ATPase Vps4 on ESCRT-III

Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Ist1, Vta1, and Did2. The order of events leading to active, ESCRT-III–associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Ist1-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in turn is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer.     

Functional Dynamics of Hexameric Helicase Probed by Hydrogen Exchange and Simulation

The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason, it is a significant challenge to investigate these assemblies using conventional experimental techniques. One of the most promising experimental techniques is hydrogen-deuterium exchange detected by mass spectrometry. Here, we describe to our knowledge a new computational method for quantitative interpretation of deuterium exchange kinetics and apply it to a hexameric viral helicase P4 that unwinds and translocates RNA into a virus capsid at the expense of ATP hydrolysis. Room-temperature dynamics probed by a hundred nanoseconds of all-atom molecular dynamics simulations is sufficient to predict the exchange kinetics of most sequence fragments and provide a residue-level interpretation of the low-resolution experimental results. The strategy presented here is also a valuable tool to validate experimental data, e.g., assignments, and to probe mechanisms that cannot be observed by x-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring.