Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4

The AAA+ ATPases are essential for various activities such as membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA ATPase Vps4, which is central to endosomal traffic to lysosomes, retroviral budding and cytokinesis, dissociates ESCRT complexes (the endosomal sorting complexes required for transport) from membranes. Here we show that, of the six ESCRT--related subunits in yeast, only Vps2 and Did2 bind the MIT (microtubule interacting and transport) domain of Vps4, and that the carboxy-terminal 30 residues of the subunits are both necessary and sufficient for interaction. We determined the crystal structure of the Vps2 C terminus in a complex with the Vps4 MIT domain, explaining the basis for selective ESCRT-III recognition. MIT helices alpha2 and alpha3 recognize a (D/E)xxLxxRLxxL(K/R) motif, and mutations within this motif cause sorting defects in yeast. Our crystal structure of the amino-terminal domain of an archaeal AAA ATPase of unknown function shows that it is closely related to the MIT domain of Vps4. The archaeal ATPase interacts with an archaeal ESCRT-III-like protein even though these organisms have no endomembrane system, suggesting that the Vps4/ESCRT-III partnership is a relic of a function that pre-dates the divergence of eukaryotes and Archaea.     

A novel brain ATPase with properties expected for the fast axonal transport motor

Identification of the ATPase involved in fast axonal transport of membranous organelles has proven difficult. Myosin and dynein, other ATPases known to be involved in cell motility, have properties that are inconsistent with the established properties of fast axonal transport, an essential component of which is readily solubilized in physiological buffer conditions rather than being stably associated with either membranous organelles or cytoskeletal elements. Adenylyl imidodiphosphate (AMP-PNP), a nonhydrolysable analogue of ATP, is a potent inhibitor of fast axonal transport that results in a stable interaction of membranous organelles with microtubules. Here we report the identification and partial characterization of an ATPase activity from brain whose binding to microtubules is stabilized by AMP-PNP. This ATPase activity seems to be associated with a polypeptide of relative molecular mass (Mr) 130,000 that is highly enriched in microtubule pellets after incubation with AMP-PNP and a soluble fraction from chick brain. This novel ATPase fraction has the predicted characteristics of the motor involved in fast axonal transport. Common features between the ATPase and fast axonal transport include interaction with the cytoskeleton in the presence of AMP-PNP, ready extractability, no Ca2+ dependence and inhibition by EDTA.     

The 2.8 Å crystal structure of the dynein motor domain

Dyneins are microtubule-based AAA(+) motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8?? resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA(+) ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.     

Cytoplasmic dynein is localized to kinetochores during mitosis

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.     

Localization of cytoplasmic dynein to mitotic spindles and kinetochores

What is the origin of the forces generating chromosome and spindle movements in mitosis? Both microtubule dynamics and microtubule-dependent motors have been proposed as the source of these motor forces. Cytoplasmic dynein and kinesin are two soluble proteins that power membranous organelle movements on microtubules. Kinesin directs movement of organelles to the 'plus' end of microtubules, and is found at the mitotic spindle in sea urchin embryos, but not in mammalian cells. Cytoplasmic dynein translocates organelles to the 'minus' end of microtubules, and is composed of two heavy chains and several light chains. We report here that monoclonal antibodies to two of these subunits and to another polypeptide that associates with dynein localize the protein to the mitotic spindle and to the kinetochores of isolated chromosomes, suggesting that cytoplasmic dynein is important in powering movements of the spindle and chromosomes in dividing cells.     

Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.     

Interaction of brain cytoplasmic dynein and MAP2 with a common sequence at the C terminus of tubulin

Two main types of microtubule-associated proteins (MAPs) have been identified in neuronal cells. The fibrous MAPs, including MAP2 and tau, serve to organize and regulate the assembly of microtubules. A second distinct class of force-producing MAPs, including kinesin, dynein and dynamin, are involved in microtubule-based movement. These proteins are mechanochemical ATPases which seem to be responsible for the bidirectional transport of organelles and perhaps also the movement of chromosomes. Here we report that MAP2 inhibits microtubule gliding on dynein-coated coverslips, as well as the microtubule-activated ATPase of dynein, indicating that MAP2 and other fibrous MAPs could be important modulators of microtubule-based motility in vivo. By proteolytic modification of tubulin, we found that dynein interacts with microtubules at the C termini of alpha- and beta-tubulin, the regions previously reported to be the sites for the interaction of MAP2. The use of site-directed antibodies implicates a small region of alpha- and beta-tubulin, containing the sequence Glu-Gly-Glu-Glu, as the site of the interaction of dynein and MAP2 with the microtubule.     

Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease

The AAA domain, a conserved Walker-type ATPase module, is a feature of members of the AAA family of proteins, which are involved in many cellular processes, including vesicular transport, organelle biogenesis, microtubule rearrangement and protein degradation. The function of the AAA domain, however, has not been explained. Membrane-anchored AAA proteases of prokaryotic and eukaryotic cells comprise a subfamily of AAA proteins that have metal-dependent peptidase activity and mediate the degradation of non-assembled membrane proteins. Inactivation of an orthologue of this protease family in humans causes neurodegeneration in hereditary spastic paraplegia. Here we investigate the AAA domain of the yeast protein Yme1, a subunit of the iota-AAA protease located in the inner membrane of mitochondria. We show that Yme1 senses the folding state of solvent-exposed domains and specifically degrades unfolded membrane proteins. Substrate recognition and binding are mediated by the amino-terminal region of the AAA domain. The purified AAA domain of Yme1 binds unfolded polypeptides and suppresses their aggregation. Our results indicate that the AAA domain of Ymel has a chaperone-like activity and suggest that the AAA domains of other AAA proteins may have a similar function.     

Identification of kinesin in sea urchin eggs, and evidence for its localization in the mitotic spindle

To understand the molecular basis of microtubule-associated motility during mitosis, the mechanochemical factors that generate the relevant motile force must be identified. Myosin, the ATPase that interacts with actin to produce the force for muscle contraction and other forms of cell motility, is believed to be involved in cytokinesis but not in mitosis. Dynein, the mechanochemical enzyme that drives microtubule sliding in eukaryotic cilia and flagella, has been identified in the cytoplasm of sea urchin eggs, but the evidence that it is involved in cytoplasmic microtubule-based motility (rather than serving as a precursor for embryonic cilia) is equivocal. Microtubule-associated ATPases have been prepared from other tissues, but their role in cytoplasmic motility is also unknown. Recent work on axoplasmic transport, however, has led to the identification of a novel mechanochemical protein called kinesin, which is thought to generate the force for moving vesicles along axonal microtubules. These results suggest that kinesin may also be a mechanochemical factor for non-axoplasmic forms of microtubule-based motility, such as mitosis. We describe here the identification and isolation of a kinesin-like protein from the cytoplasm of sea urchin eggs. We present evidence that this protein is localized in the mitotic spindle, and propose that it may be a mechanochemical factor for some form of motility associated with the mitotic spindle.     

虎纹蛙病毒的ATP酶基因克隆和序列分析

利用对应于蛙病毒—3型(FV3）ATP酶基因(ATPase)的162—178nt和1186—1174ntt碱基序列作为引物，采用PCR方法扩增得到虎纹蛙病毒(RTV)的ATP酶基因，并对该基因进行克隆、测序和分析。基因读码框大小为945hp，预计可编码一相对分子质量为35500的蛋白质。氨基酸序列比较结果，RTV的ATPase基因与虹彩病毒科蛙病毒属的代表种FV3的一致性最高，为87．9％，与该科其它脊椎动物病毒的一致性在50％-52％之间。ATP酶蛋白质结构域分析可知该基因编码的ATP酶含有Walker型ATP酶AAA族蛋白质的全部结构域，其中既具有Walker型ATP酶所具有的Walker a和Walker b保守区，还含有AAA族蛋白质基因所具有的SRH高度保守区，因此，该基因是一完整的活性蛋白编码基因。     

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.     

Active sliding between cytoplasmic microtubules

Microtubules are versatile cellular polymers that play a role in cell shape determination and mediate various motile processes such as ciliary and flagellar bending, chromosome movements and organelle transport. That a sliding microtubule mechanism can generate force has been demonstrated in highly ordered structures such as axonemes, and microtubule-based force generation almost certainly contributes to the function of mitotic and meiotic spindles. Most cytoplasmic microtubule arrays, however, do not exhibit the structural regularity of axonemes and some spindles, and often appear disorganized. Yet many cellular activities (such as shape changes during morphogenesis, axonal extension and spindle assembly) involve highly coordinated microtubule behaviour and possibly require force generated by an intermicrotubule sliding mechanism, or perhaps use sliding to move microtubules rapidly into a protrusion for stabilization. Here we show that active sliding between cytoplasmic microtubules can occur in microtubule bundles of the amoeba Reticulomyxa. A force-producing mechanism of this sort could be used by this organism to facilitate the extension of cell processes and to generate the dynamic movements of the cytoplasmic network.     

Four ATP-binding sites in the midregion of the beta heavy chain of dynein.

The 'motor' proteins of eukaryotic cells contain specialized domains that hydrolyse ATP to produce force and movement along a cytoskeletal polymer (actin in the case of the myosin family; microtubules in the case of the kinesin family and dyneins). There are motor-protein superfamilies in which each member has a conserved force-generating domain joined to a different 'tail' which conveys specific attachment properties. The minus-end-directed microtubule motors, the dyneins, may also constitute a superfamily of force-generating proteins with distinct attachment domains. Axonemal outer-arm dynein from sea urchin spermatozoa is a multimeric protein consisting of two heavy chains (alpha and beta) with ATPase activity, three intermediate chains and several light chains. Here I report the sequence of cloned complementary DNA encoding the beta heavy chain of a dynein motor molecule. The predicted amino-acid sequence reveals four ATP-binding consensus sequences in the central domain. The dynein beta heavy chain is thought to associate transiently with a microtubule during ATP hydrolysis, but the ATP-dependent microtubule-binding sequence common to the kinesin superfamily is not found in the dynein beta heavy chain. These unique features distinguish the dynein beta heavy chain from other motor protein superfamilies and may be characteristic of the dynein superfamily.     

Dynein structure and power stroke

Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke.     

Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein

Dynein, an ATPase, is the force-generating protein in cilia and flagella. It has long been speculated that cytoplasmic microtubules contain a related enzyme involved in cell division or in intracellular organelle transport. A 'cytoplasmic dynein' has been described in sea urchin eggs, but because the egg stockpiles precursors for both cytoplasmic and ciliary microtubules, the role of this enzyme in the cell has remained unresolved. We recently found that the microtubule-associated protein (MAP) 1C (ref. 6) from brain is a microtubule-activated ATPase that produces force in the direction corresponding to retrograde organelle transport in the cell. MAP 1C has several similar properties to ciliary and flagellar dynein. Here we show directly, using scanning transmission electron microscopy, that MAP 1C is structurally equivalent to the ciliary and flagellar enzyme and is the long-sought cytoplasmic analogue of this enzyme.     

Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-? crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.     

Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast

Microtubules are essential intracellular structures involved in several cellular phenomena, including polarity establishment and chromosome segregation. Because the nuclear envelope persists during mitosis (closed mitosis) in fission yeast (Schizosaccharomyces pombe), cytoplasmic microtubules must be reorganized into the spindle in the compartmentalized nucleus on mitotic entry. An ideal mechanism might be to take advantage of an evolutionarily conserved microtubule formation system that uses the Ran-GTPase nuclear transport machinery, but no targets of Ran for spindle formation have been identified in yeast. Here we show that a microtubule-associated protein, Alp7, which forms a complex with Alp14, is a target of Ran in yeast for spindle formation. The Ran-deficient pim1 mutant (pim1-F201S) failed to show mitosis-specific nuclear accumulation of Alp7. Moreover, this mutant exhibited compromised spindle formation and early mitotic delay. Importantly, these defects were suppressed by Alp7 that was artificially targeted to the nucleus by a Ran-independent and importin-alpha-mediated system. Thus, Ran targets Alp7-Alp14 to achieve nuclear spindle formation, and might differentiate its targets depending on whether the organism undergoes closed or open mitosis.     

DNA stretching by bacterial initiators promotes replication origin opening

Many replication initiators form higher-order oligomers that process host replication origins to promote replisome formation. In addition to dedicated duplex-DNA-binding domains, cellular initiators possess AAA+ (ATPases associated with various cellular activities) elements that drive functions ranging from protein assembly to origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been proposed to assist in single-stranded DNA formation during origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of Aquifex aeolicus DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA opens replication origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal notable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic-acid-dependent, AAA+ systems.     

Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin

Spastin, the most common locus for mutations in hereditary spastic paraplegias, and katanin are related microtubule-severing AAA ATPases involved in constructing neuronal and non-centrosomal microtubule arrays and in segregating chromosomes. The mechanism by which spastin and katanin break and destabilize microtubules is unknown, in part owing to the lack of structural information on these enzymes. Here we report the X-ray crystal structure of the Drosophila spastin AAA domain and provide a model for the active spastin hexamer generated using small-angle X-ray scattering combined with atomic docking. The spastin hexamer forms a ring with a prominent central pore and six radiating arms that may dock onto the microtubule. Helices unique to the microtubule-severing AAA ATPases surround the entrances to the pore on either side of the ring, and three highly conserved loops line the pore lumen. Mutagenesis reveals essential roles for these structural elements in the severing reaction. Peptide and antibody inhibition experiments further show that spastin may dismantle microtubules by recognizing specific features in the carboxy-terminal tail of tubulin. Collectively, our data support a model in which spastin pulls the C terminus of tubulin through its central pore, generating a mechanical force that destabilizes tubulin-tubulin interactions within the microtubule lattice. Our work also provides insights into the structural defects in spastin that arise from mutations identified in hereditary spastic paraplegia patients.     

Inner-arm dynein c of Chlamydomonas flagella is a single-headed processive motor.

Axonemal dyneins are force-generating ATPases that produce movement of eukaryotic cilia and flagella. Several studies indicate that inner-arm dyneins mainly produce bending moments in flagella and that these motors have inherent oscillations in force and motility. Processive motors such as kinesins have high duty ratios of attached to total ATPase cycle (attached plus detached) times compared to sliding motors such as myosin. Here we provide evidence that subspecies-c, a single-headed axonemal inner-arm dynein, is processive but has a low duty ratio. Ultrastructurally it is similar to other dyneins, with a single globular head, long stem and a slender stalk that attaches to microtubules. In vitro studies of microtubules sliding over surfaces coated with subspecies-c at low densities (measured by single-molecule fluorescence) show that a single molecule is sufficient to move a microtubule more than 1 microm at 0.7 microm s(-1). When many motors interact the velocity is 5.1 microm s(-1), fitting a duty ratio of 0.14. Using optical trap nanometry, we show that beads carrying a single subspecies-c motor move processively along the microtubules in 8-nm steps but slip backwards under high loads. These results indicate that dynein subspecies-c functions in a very different way from conventional motor proteins, and has properties that could produce self-oscillation in vivo.