AAA ( A TPases A ssociated with diverse cellular A ctivities ) proteins (speak: triple-A ATPases) are a large group of protein family sharing a common conserved module of approximately 230 amino acid residues. This is a large, functionally diverse protein family belonging to the AAA+ protein superfamily of ring-shaped P-loop NTPases , which exert their activity through the energy-dependent remodeling or translocation of macromolecules.
50-613: AAA proteins couple chemical energy provided by ATP hydrolysis to conformational changes which are transduced into mechanical force exerted on a macromolecular substrate. AAA proteins are functionally and organizationally diverse, and vary in activity, stability, and mechanism. Members of the AAA family are found in all organisms and they are essential for many cellular functions. They are involved in processes such as DNA replication , protein degradation , membrane fusion , microtubule severing , peroxisome biogenesis , signal transduction and
100-460: A chemical signal that punts the dynein to the other side of the cell. It does this repeatedly so the chromosomes end up in the center of the cell, which is necessary in mitosis. Budding yeast have been a powerful model organism to study this process and has shown that dynein is targeted to plus ends of astral microtubules and delivered to the cell cortex via an offloading mechanism. Dynein and kinesin can both be exploited by viruses to mediate
150-545: A coordinated fashion so that the cilia or flagella can move back and forth. The radial spoke has been proposed as the (or one of the) structures that synchronizes this movement. The regulation of axonemal dynein activity is critical for flagellar beat frequency and cilia waveform. Modes of axonemal dynein regulation include phosphorylation, redox, and calcium. Mechanical forces on the axoneme also affect axonemal dynein function. The heavy chains of inner and outer arms of axonemal dynein are phosphorylated/dephosphorylated to control
200-502: A major binding site that co-opts dynein. Each molecule of the dynein motor is a complex protein assembly composed of many smaller polypeptide subunits. Cytoplasmic and axonemal dynein contain some of the same components, but they also contain some unique subunits. Cytoplasmic dynein, which has a molecular mass of about 1.5 megadaltons (MDa), is a dimer of dimers, containing approximately twelve polypeptide subunits: two identical "heavy chains", 520 kDa in mass, which contain
250-477: A normal complement of chromosomes. The formation of chiasmata (crossover recombination events) appears to generally facilitate proper segregation. However, in the fission yeast Schizosaccharomyces pombe , when chiasmata are absent, dynein promotes segregation. Dhc1, the motor subunit of dynein, is required for chromosomal segregation in both the presence and absence of chiasmata. The dynein light chain Dlc1 protein
300-470: A ring-shaped structure with a central pore. These proteins produce a molecular motor that couples ATP binding and hydrolysis to changes in conformational states that can be propagated through the assembly in order to act upon a target substrate, either translocating or remodelling the substrate. The central pore may be involved in substrate processing. In the hexameric configuration, the ATP-binding site
350-420: Is a AAA-type ATPase involved in this MVB sorting pathway. It had originally been identified as a ”class E” vps (vacuolar protein sorting) mutant and was subsequently shown to catalyse the dissociation of ESCRT complexes. Vps4p is anchored via Vps46p to the endosomal membrane. Vps4p assembly is assisted by the conserved Vta1p protein, which regulates its oligomerization status and ATPase activity. AAA proteases use
400-451: Is a highly exergonic process. The amount of released energy depends on the conditions in a particular cell. Specifically, the energy released is dependent on concentrations of ATP, ADP and P i . As the concentrations of these molecules deviate from values at equilibrium, the value of Gibbs free energy change (Δ G ) will be increasingly different. In standard conditions (ATP, ADP and P i concentrations are equal to 1M, water concentration
450-449: Is a member of the Ras superfamily , mediates the attachment of several cargo adaptors to the dynein motor. The other tail subunits may also help facilitate this interaction as evidenced in a low resolution structure of dynein-dynactin-BicD2. One major form of motor regulation within cells for dynein is dynactin. It may be required for almost all cytoplasmic dynein functions. Currently, it is
500-450: Is affected. AAA proteins are involved in protein degradation , membrane fusion , DNA replication , microtubule dynamics, intracellular transport, transcriptional activation, protein refolding, disassembly of protein complexes and protein aggregates . Dyneins , one of the three major classes of motor protein , are AAA proteins which couple their ATPase activity to molecular motion along microtubules . The AAA-type ATPase Cdc48p/p97
550-590: Is almost twice as much as the energy produced under standard conditions. Dynein Dyneins are a family of cytoskeletal motor proteins that move along microtubules in cells . They convert the chemical energy stored in ATP to mechanical work . Dynein transports various cellular cargos , provides forces and displacements important in mitosis , and drives the beat of eukaryotic cilia and flagella . All of these functions rely on dynein's ability to move towards
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#1732802056032600-466: Is equal to 55 M) the value of Δ G is between -28 and -34 kJ/mol. The range of the Δ G value exists because this reaction is dependent on the concentration of Mg cations, which stabilize the ATP molecule. The cellular environment also contributes to differences in the Δ G value since ATP hydrolysis is dependent not only on the studied cell, but also on the surrounding tissue and even the compartment within
650-482: Is greatly stabilized by multiple resonance structures , making the products (ADP and P i ) lower in energy than the reactant (ATP). The high negative charge density associated with the three adjacent phosphate units of ATP also destabilizes the molecule, making it higher in energy. Hydrolysis relieves some of these electrostatic repulsions, liberating useful energy in the process by causing conformational changes in enzyme structure. In humans, approximately 60 percent of
700-437: Is much greater than the standard value. The nonstandard conditions of the cell actually result in a more favorable reaction. In one particular study, to determine Δ G in vivo in humans, the concentration of ATP, ADP, and P i was measured using nuclear magnetic resonance. In human muscle cells at rest, the concentration of ATP was found to be around 4 mM and the concentration of ADP was around 9 μM. Inputing these values into
750-592: Is perhaps the best-studied AAA protein. Misfolded secretory proteins are exported from the endoplasmic reticulum (ER) and degraded by the ER-associated degradation pathway ( ERAD ). Nonfunctional membrane and luminal proteins are extracted from the ER and degraded in the cytosol by proteasomes. Substrate retrotranslocation and extraction is assisted by the Cdc48p(Ufd1p/Npl4p) complex on the cytosolic side of
800-658: Is positioned at the interface between the subunits. Upon ATP binding and hydrolysis, AAA enzymes undergo conformational changes in the AAA-domains as well as in the N-domains. These motions can be transmitted to substrate protein. ATP hydrolysis by AAA ATPases is proposed to involve nucleophilic attack on the ATP gamma-phosphate by an activated water molecule, leading to movement of the N-terminal and C-terminal AAA subdomains relative to each other. This movement allows
850-474: Is released by the hydrolysis of ATP. However, when the P-O bonds are broken, input of energy is required. It is the formation of new bonds and lower-energy inorganic phosphate with a release of a larger amount of energy that lowers the total energy of the system and makes it more stable. Hydrolysis of the phosphate groups in ATP is especially exergonic , because the resulting inorganic phosphate molecular ion
900-501: Is the catabolic reaction process by which chemical energy that has been stored in the high-energy phosphoanhydride bonds in adenosine triphosphate (ATP) is released after splitting these bonds, for example in muscles , by producing work in the form of mechanical energy . The product is adenosine diphosphate (ADP) and an inorganic phosphate (P i ). ADP can be further hydrolyzed to give energy, adenosine monophosphate (AMP), and another inorganic phosphate (P i ). ATP hydrolysis
950-443: Is the final link between the energy derived from food or sunlight and useful work such as muscle contraction , the establishment of electrochemical gradients across membranes, and biosynthetic processes necessary to maintain life. Anhydridic bonds are often labelled as " high-energy bonds" . P-O bonds are in fact fairly strong (~30 kJ/mol stronger than C-N bonds) and themselves not particularly easy to break. As noted below, energy
1000-499: Is the largest. There is no definitive evidence that dynactin by itself affects the velocity of the motor. It does, however, affect the processivity of the motor. The binding regulation is likely allosteric: experiments have shown that the enhancements provided in the processivity of the dynein motor do not depend on the p150 subunit binding domain to the microtubules. Axonemal dyneins come in multiple forms that contain either one, two or three non-identical heavy chains (depending upon
1050-561: Is thought to occur through unfolded protein domains in the substrate protein. In HslU, a bacterial ClpX/ClpY homologue of the HSP100 family of AAA proteins, the N- and C-terminal subdomains move towards each other when nucleotides are bound and hydrolysed. The terminal domains are most distant in the nucleotide-free state and closest in the ADP-bound state. Thereby the opening of the central cavity
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#17328020560321100-399: Is well established as the primary site of ATP hydrolysis in dynein. When ATP binds to AAA1, it initiates a conformational change of the AAA domain ring into the “closed” configuration, movement of the buttress, and a conformational change in the linker. The linker becomes bent and shifts from AAA5 to AAA2 while remaining bound to AAA1. One attached alpha -helix from the stalk is pulled by
1150-490: The ATPase activity and are thus responsible for generating movement along the microtubule; two 74 kDa intermediate chains which are believed to anchor the dynein to its cargo; two 53–59 kDa light intermediate chains; and several light chains. The force-generating ATPase activity of each dynein heavy chain is located in its large doughnut-shaped "head", which is related to other AAA proteins , while two projections from
1200-407: The axonemes of cilia and flagella and is found only in cells that have those structures. Cytoplasmic dynein, found in all animal cells and possibly plant cells as well, performs functions necessary for cell survival such as organelle transport and centrosome assembly. Cytoplasmic dynein moves processively along the microtubule; that is, one or the other of its stalks is always attached to
1250-429: The mitotic spindles for cell division. Dynein carries organelles, vesicles and possibly microtubule fragments along the axons of neurons toward the cell body in a process called retrograde axonal transport . Additionally, dynein motor is also responsible for the transport of degradative endosomes retrogradely in the dendrites. Cytoplasmic dynein positions the spindle at the site of cytokinesis by anchoring to
1300-558: The regulation of gene expression . The AAA proteins contain two domains, an N-terminal alpha/beta domain that binds and hydrolyzes nucleotides (a Rossmann fold ) and a C-terminal alpha-helical domain. The N-terminal domain is 200-250 amino acids long and contains Walker A and Walker B motifs , and is shared in common with other P-loop NTPases, the superfamily which includes the AAA family. Most AAA proteins have additional domains that are used for oligomerization , substrate binding and/or regulation. These domains can lie N- or C-terminal to
1350-669: The AAA module. Some classes of AAA proteins have an N-terminal non-ATPase domain which is followed by either one or two AAA domains (D1 and D2). In some proteins with two AAA domains, both are evolutionarily well conserved (like in Cdc48/p97 ). In others, either the D2 domain (like in Pex1p and Pex6p) or the D1 domain (in Sec18p/NSF) is better conserved in evolution. While the classical AAA family
1400-510: The MT, triggering the power stroke. The linker returns to a straight conformation and swings back to AAA5 from AAA2 and creates a lever-action, producing the greatest displacement of dynein achieved by the power stroke The cycle concludes with the release of ADP, which returns the AAA domain ring back to the “open” configuration. Yeast dynein can walk along microtubules without detaching, however in metazoans, cytoplasmic dynein must be activated by
1450-424: The above equations yields Δ G = -64 kJ/mol. After ischemia , when the muscle is recovering from exercise, the concentration of ATP is as low as 1 mM and the concentration of ADP is around 7 μM. Therefore, the absolute Δ G would be as high as -69 kJ/mol. By comparing the standard value of Δ G and the experimental value of Δ G , one can see that the energy released from the hydrolysis of ATP, as measured in humans,
1500-453: The best studied dynein partner. Dynactin is a protein that aids in intracellular transport throughout the cell by linking to cytoplasmic dynein. Dynactin can function as a scaffold for other proteins to bind to. It also functions as a recruiting factor that localizes dynein to where it should be. There is also some evidence suggesting that it may regulate kinesin-2. The dynactin complex is composed of more than 20 subunits, of which p150(Glued)
1550-421: The binding of dynactin , another multisubunit protein that is essential for mitosis , and a cargo adaptor. The tri-complex, which includes dynein, dynactin and a cargo adaptor, is ultra-processive and can walk long distances without detaching in order to reach the cargo's intracellular destination. Cargo adaptors identified thus far include BicD2 , Hook3 , FIP3 and Spindly. The light intermediate chain, which
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1600-453: The buttress, sliding the helix half a heptad repeat relative to its coilled-coil partner, and kinking the stalk. As a result, the MTBD of dynein enters a low-affinity state, allowing the motor to move to new binding sites. Following hydrolysis of ATP, the stalk rotates, moving dynein further along the MT. Upon the release of the phosphate, the MTBD returns to a high affinity state and rebinds
1650-423: The cell cortex and pulling on astral microtubules emanating from centrosome . While a postdoctoral student at MIT, Tomomi Kiyomitsu discovered how dynein has a role as a motor protein in aligning the chromosomes in the middle of the cell during the metaphase of mitosis. Dynein pulls the microtubules and chromosomes to one end of the cell. When the end of the microtubules become close to the cell membrane, they release
1700-505: The cell. Variability in the Δ G values is therefore to be expected. The relationship between the standard Gibbs free energy change Δ r G and chemical equilibrium is revealing. This relationship is defined by the equation Δ r G = - RT ln( K ), where K is the equilibrium constant , which is equal to the reaction quotient Q in equilibrium. The standard value of Δ G for this reaction is, as mentioned, between -28 and -34 kJ/mol; however, experimentally determined concentrations of
1750-582: The ciliary axoneme. During the "power stroke", which causes movement, the AAA ATPase motor domain undergoes a conformational change that causes the microtubule-binding stalk to pivot relative to the cargo-binding tail with the result that one microtubule slides relative to the other (Karp, 2005). This sliding produces the bending movement needed for cilia to beat and propel the cell or other particles. Groups of dynein molecules responsible for movement in opposite directions are probably activated and inactivated in
1800-403: The complete cytoplasmic dynein motor enables a single dynein molecule to transport its cargo by "walking" a considerable distance along a microtubule without becoming completely detached. In the apo-state of dynein, the motor is nucleotide free, the AAA domain ring exists in an open conformation, and the MTBD exists in a high affinity state. Much about the AAA domains remains unknown, but AAA1
1850-458: The concentrations are more appropriately measured in mM, which is smaller than M by three orders of magnitude. Using these nonstandard concentrations, the calculated value of Q is much less than one. By relating Q to Δ G using the equation Δ G = Δ r G + RT ln( Q ), where Δ r G is the standard change in Gibbs free energy for the hydrolysis of ATP, it is found that the magnitude of Δ G
1900-855: The energy from ATP hydrolysis to translocate a protein inside the proteasome for degradation. AFG3L2 ; ATAD1 ; ATAD2 ; ATAD2B ; ATAD3A ; ATAD3B ; ATAD3C ; ATAD5 ; BCS1L ; CHTF18 ; CLBP ; CLPP ; CLPX ; FIGN ; FIGNL1 ; FIGNL2 ; IQCA1 ; KATNA1 ; KATNAL1 ; KATNAL2 ; LONP1 ; LONP2 ; MDN1 ; NSF ; NVL ; ORC1 ; ORC4 ; PEX1 ; PEX6 ; PSMC1 ; PSMC2 (Nbla10058); PSMC3 ; PSMC4 ; PSMC5 ; PSMC6 ; RFC1 ; RFC2 ; RFC3 ; RFC4 ; RFC5 ; RUVBL1 ; RUVBL2 ; SPAST ; SPATA5 (SPAF); SPATA5L1 ; SPG7 ; TRIP13 ; VCP ; VPS4A ; VPS4B ; WRNIP1 ; YME1L1 (FTSH); TOR1A ; TOR1B ; TOR2A ; TOR3A ; TOR4A ; AK6 (CINAP); CDC6 ; AFG3L1P; ATP hydrolysis ATP hydrolysis
1950-418: The energy released from the hydrolysis of ATP produces metabolic heat rather than fuel the actual reactions taking place. Due to the acid-base properties of ATP, ADP, and inorganic phosphate, the hydrolysis of ATP has the effect of lowering the pH of the reaction medium. Under certain conditions, high levels of ATP hydrolysis can contribute to lactic acidosis . Hydrolysis of the terminal phosphoanhydridic bond
2000-535: The exertion of mechanical force, amplified by other ATPase domains within the same oligomeric structure. The additional domains in the protein allow for regulation or direction of the force towards different goals. AAA proteins are not restricted to eukaryotes . Prokaryotes have AAA which combine chaperone with proteolytic activity, for example in ClpAPS complex, which mediates protein degradation and recognition in E. coli . The basic recognition of proteins by AAAs
2050-406: The head connect it to other cytoplasmic structures. One projection, the coiled-coil stalk, binds to and "walks" along the surface of the microtubule via a repeated cycle of detachment and reattachment. The other projection, the extended tail, binds to the light intermediate, intermediate and light chain subunits which attach dynein to its cargo. The alternating activity of the paired heavy chains in
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2100-414: The involved molecules reveal that the reaction is not at equilibrium. Given this fact, a comparison between the equilibrium constant, K , and the reaction quotient, Q , provides insight. K takes into consideration reactions taking place in standard conditions, but in the cellular environment the concentrations of the involved molecules (namely, ATP, ADP, and P i ) are far from the standard 1 M. In fact,
2150-446: The membrane. On the cytosolic side, the substrate is ubiquitinated by ER-based E2 and E3 enzymes before degradation by the 26S proteasome. Multivesicular bodies are endosomal compartments that sort ubiquitinated membrane proteins by incorporating them into vesicles. This process involves the sequential action of three multiprotein complexes, ESCRT I to III ( ESCRT standing for 'endosomal sorting complexes required for transport'). Vps4p
2200-477: The microtubule so that the dynein can "walk" a considerable distance along a microtubule without detaching. Cytoplasmic dynein helps to position the Golgi complex and other organelles in the cell. It also helps transport cargo needed for cell function such as vesicles made by the endoplasmic reticulum , endosomes , and lysosomes (Karp, 2005). Dynein is involved in the movement of chromosomes and positioning
2250-436: The minus-end of the microtubules, known as retrograde transport ; thus, they are called "minus-end directed motors". In contrast, most kinesin motor proteins move toward the microtubules' plus-end, in what is called anterograde transport . Dyneins can be divided into two groups: cytoplasmic dyneins and axonemal dyneins , which are also called ciliary or flagellar dyneins. Axonemal dynein causes sliding of microtubules in
2300-408: The organism and location in the cilium ). Each heavy chain has a globular motor domain with a doughnut-shaped structure believed to resemble that of other AAA proteins , a coiled coil "stalk" that binds to the microtubule, and an extended tail (or "stem") that attaches to a neighboring microtubule of the same axoneme . Each dynein molecule thus forms a cross-bridge between two adjacent microtubules of
2350-444: The rate of microtubule sliding. Thioredoxins associated with the other axonemal dynein arms are oxidized/reduced to regulate where dynein binds in the axoneme. Centerin and components of the outer axonemal dynein arms detect fluctuations in calcium concentration. Calcium fluctuations play an important role in altering cilia waveform and flagellar beat frequency (King, 2012). The protein responsible for movement of cilia and flagella
2400-507: The viral replication process. Many viruses use the microtubule transport system to transport nucleic acid/protein cores to intracellular replication sites after invasion host the cell membrane. Not much is known about virus' motor-specific binding sites, but it is known that some viruses contain proline-rich sequences (that diverge between viruses) which, when removed, reduces dynactin binding, axon transport (in culture), and neuroinvasion in vivo. This suggests that proline-rich sequences may be
2450-424: Was based on motifs, the family has been expanded using structural information and is now termed the AAA family. AAA proteins are divided into seven basic clades , based on secondary structure elements included within or near the core AAA fold: clamp loader, initiator, classic, superfamily III helicase, HCLR, H2-insert, and PS-II insert. AAA ATPases assemble into oligomeric assemblies (often homo-hexamers) that form
2500-409: Was first discovered and named dynein in 1963 (Karp, 2005). 20 years later, cytoplasmic dynein, which had been suspected to exist since the discovery of flagellar dynein, was isolated and identified (Karp, 2005). Segregation of homologous chromosomes to opposite poles of the cell occurs during the first division of meiosis . Proper segregation is essential for producing haploid meiotic products with
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