Available online at www.sciencedirect.com
ScienceDirect The unique enzymatic and mechanistic properties of plant myosins Arnon Henn1 and Einat Sadot2 Myosins are molecular motors that move along actin-filament tracks. Plants express two main classes of myosins, myosin VIII and myosin XI. Along with their relatively conserved sequence and functions, plant myosins have acquired some unique features. Myosin VIII has the enzymatic characteristics of a tension sensor and/or a tension generator, similar to functions found in other eukaryotes. Interestingly, class XI plant myosins have gained a novel function that consists of propelling the exceptionally rapid cytoplasmic streaming. This specific class includes the fastest known translocating molecular motors, which can reach an extremely high velocity of about 60 mm s1. However, the enzymatic properties and mechanistic basis for these remarkable manifestations are not yet fully understood. Here we review recent progress in understanding the uniqueness of plant myosins, while emphasizing the unanswered questions. Addresses 1 Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel 2 The Institute of Plant Sciences, Volcani Center, PO Box 6, Bet-Dagan 5025000, Israel Corresponding authors: Henn, Arnon ([email protected]) and Sadot, Einat ([email protected])
Current Opinion in Plant Biology 2014, 22:65–70 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´
http://dx.doi.org/10.1016/j.pbi.2014.09.006 1369-5266/# 2014 Elsevier Ltd. All rights reserved.
Introduction Live-cell imaging of plant cells reveals strikingly rapid movement of organelles and vesicles, rearrangement of the endoplasmic reticulum (ER) and constant remodeling of the tonoplast. Under certain conditions slower movement of the chloroplasts and nucleus can also be observed. This intracellular dynamic is believed to enable the immotile plant to respond quickly to abiotic and biotic stresses. The machinery underlying these vigorous movements involves actin-based molecular motors, the myosins and actin microfilaments that together drive the formation of streaming patterns in plant cells. Myosins utilize ATP to perform mechanical work such as muscle contraction and www.sciencedirect.com
the translocation of cargo along actin filaments in eukaryotic cells [1]. Across all eukaryotes there are 35 known classes of myosins [2]. In plants there are two main classes, VIII, XI [3], which are widely distributed in higher and lower plants. Plant myosins are involved in various organelles’ motility [4,5,6–14], ER remodeling [15–18] and cytoplasmic streaming [19,20,21,22]. They have been found in the plasmodesmata [23–25,26] and can be involved in targeted RNA transport [27], viral spread from cell to cell [28–31] and in a plant’s size and shape determination [6,22,32]. All known myosins share a highly conserved amino-terminal motor domain followed by a neck (also referred to as the ‘lever arm’) and the tail regions. The neck region, which contains conserved IQ repeats (as many as one to six in tandem), serves as the light chains binding domains and the tail region which displays diverse interacting domains such as cargo and membrane binding motifs [1]. The myosin actin-dependent ATPase cycle includes at least six nucleotide-linked, biochemical intermediates (Figure 1a). The cycling of myosin on actin is driven by ATP binding, hydrolysis and product release, and can be described as follows (Figure 1a from left to right): ATP binding to actomyosin causes myosin head to dissociate from actin. Detached myosin hydrolyzes ATP to ADP and Pi, which drives the repriming of the lever arm to the ‘prepower stroke’ state. This post hydrolysis state can rebind actin with a higher affinity than before the hydrolysis of ATP. Upon the rebinding of myosin-ADP-Pi state to actin conformational changes within the myosin motor domain are coupled to the release of Pi and the lever arm rotation relative to the actin-bound myosin head. This lever arm rotation transition is the force-generating step, or the ‘power-stroke’, of the myosin ATPase cycle, which is next followed by ADP release, and the formation of the myosinactin nucleotide free state. Thereafter, the catalytic cycle begins again with ATP binding. Specific nucleotide states dictate the affinity of the myosin motor domain towards actin. The strength of the myosin nucleotide binding states towards actin follows from strong to weak binding affinities as such: actomyosin > actomyosin-ADP > actomyosinADP-Pi > actomyosin-ATP. Although this ATPase cycle kinetic pathway is highly conserved among all myosins studied to date, the enzymatic properties of the catalytic motor domain display very large deviations in their kinetic and thermodynamic properties, that is, each myosin isoform displays different rate and equilibrium constants for the biochemical transitions described in Figure 1a [33,34]. These different rate and equilibrium constants give raise to different ATPase cycling-times, affinities toward actin and motility rates among myosins. They are the hallmarks Current Opinion in Plant Biology 2014, 22:65–70
66 Cell biology
Figure 1
(a)
Myosin XIChara: Very fast ADP release
“Weakly Bound”
“Strongly Bound” AM + ATP
K’H
AM ATP
KA
“Strongly Bound”
AM ADP Pi
K’Pi
M + ATP
M ATP
KH
AM + ADP
AM ADP KAD
KAPi KT
Myosin V, VIII: Rate limiting Step during ATPase cycling
M ADP Pi
“Dissociated States” (b)
KPi
KA
M ADP
KD
M + ADP
“Thermodynamic Coupling box” Low TC for Myosin V, VIII High TC for Myosin XIChara.
Second strongly bound actomyosin-ADP state
“Strongly AM ADP
K’D1
AM
Bound” ADP’
K’D2
AM + ADP
Slow isomerization Current Opinion in Plant Biology
The actomyosin ATPase cycle. (a) Schematic representation of myosin’s conserved chemomechanical energy transduction pathway is shown. The reaction pathway in the black frame is the predominant pathway for the myosin ATPase cycle as described in the text. Key features in plant myosins VIII and XIChara in relation to the ATPase reaction scheme are shown. The dashed blue frame contains the key parameters for determining thermodynamic coupling (TC) between actin and ADP affinities toward myosin. Black and blue callouts mark unique features among measured parameters of plant myosins. Blue ovals highlight extremely rapid rate constants for Chara myosin XI. Red ovals mark rate and equilibrium constants that have not been measured for any plant myosin. (b) Expansion of the minimal kinetic scheme showing the transition from actomyosin-ADP to actomyosin with additional intermediate ADP state namely actomyosin-ADP0 indicative of tension-sensor myosins.
of the diverse enzymatic behavior among the myosin family classes and isoforms. In turns, these unique features enables each myosin isoform to be enzymatically adapted to its cellular functions. Here we describe the findings related to the motor properties of the two main classes of plant myosins and discuss their enzymatic adaptation to the relevant unique cellular functions in plants.
Class VIII myosins The first plant myosin to be cloned and sequenced was Arabidopsis thaliana myosin 1 (ATM1). Phylogenetic analysis based on its motor domain placed ATM1 in a new myosin class VIII [35]. Specific antibodies against its tail domain revealed its subcellular localization in the plasmodesmata and newly formed cell plates in the roots of maize and cress (Lepidium sativum) [36]. Subsequent green fluorescent protein (GFP) fusions confirmed these observations [25,26,37], and also suggested its localization in ER junctions [25]. Analysis of the knockout plant of all five myosin VIII transcripts of P. patens revealed that this class has a role in development and hormone Current Opinion in Plant Biology 2014, 22:65–70
homeostasis [32]. Recent detailed transient kinetic and thermodynamic studies of myosin VIII have revealed its enzymatic properties [26]. It was found that ADP release (at physiological levels of Mg2+) [26] is the rate-limiting step (the slowest transition within one complete ATPaes cycle, Figure 1a) in myosin VIII’s ATPase cycle, which contributes to the long-lived and stronglybound actomyosin-ADP state during the ATPase cycle and to high duty ratio (DR — see below). The cycling time is defined as a single complete cycle of ATP binding, hydrolysis and product release (Figure 1a). Myosin VIII displays a relatively slow to moderate ATPase cycle, with a slow motility rate that is highly regulated by Mg2+ [26]. The latter characterize local tension-bearing myosins rather than fast moving transporters [33,38]. In addition, the two states, with fast and slow dissociation rates (Figure 1b), observed in myosin VIII [26] suggest that additional work can be achieved with the slower step of ADP dissociation [38]. This is a unique feature that is found in several tension-sensor myosins [33]. Furthermore, myosin VIII displays a very weak thermodynamic www.sciencedirect.com
Plant myosins Henn and Sadot 67
coupling (TC) ratio of 2.3 [26]. TC is a measure of how the affinity of myosin to actin is affected by its binding to ADP, in comparison to how its binding to ADP is affected by actin. This simply reflects the degree of communication between the nucleotide and actin binding sites, and is derived from the free energy detailed balance of the ADP states. Detailed balance requires that in the absence of external energy input or consumption the product of the four equilibrium constants equal unity and, therefore, K 0D =K D ¼ K A =K AD (Figure 1a). Tensionsensor myosins have been shown to display very weak TC or a low ratio close to unity between the equilibrium constants [33,38]. A weak TC permits strong binding to actin in the presence of ADP, and hence to bear tension in the strongly bound ADP states. Lastly, the kinetic adaptation of plant myosin VIII for its function also stems from its measured high DR of 0.9 [26]. The DR is defined as the fraction of time that myosin spends strongly bound to actin during the complete ATPase cycle. Thus, the detailed enzymological studies of myosin VIII strongly suggest that it is enzymatically adapted to maintaining tension, such as that required for ER tethering to the plasma membrane for proper plasmodesmatal function [39]. However, the latter assumption requires in-vivo validations.
Class XI myosins Class XI myosins from Chara corallina are the fastest actin-based motors characterized to date, even faster than myosin XI members in higher plants. Class XI myosins show sequence and structural similarities to vertebrates’ transporter class V myosins [40]. Both contain a long lever arm composed of six IQ motifs, indicative of a large step size, and a coiled-coil motif, which favors a doubleheaded (dimer) myosins’ oligomeric state [41]. Despite these similarities to myosin class V, the velocity measurements by in vitro motility assay for plant myosins have demonstrated remarkably higher rates. For purified C. corallina myosin XI, the measured velocities range from 20 mm s1 up to 60 mm s1 [42–44], and for myosin XI from Nicotiana tabacum and Arabidopsis this is about 7 mm s1. These measurements are up to 150-folds and 20-folds respectively, faster than myosin V [45]. Purified myosin XI from N. tabacum was found to have a step size of 35 nm [46]. This step size was similar to that measured for myosin V, which has an identical lever arm length of six IQ motifs [45]. The step size measured for the faster Chara myosin XI was 19 nm [44]. The extraordinarily high velocity of myosin XI does not result from an unusually large step size. Calculations combining velocity with step size suggested that the ATPase cycling time for the slower measured velocity (myosin XI from N. tabacum and Arabidopsis) should be 200 ATP s1 h1 (s1 h1 is define as moles of ATP hydrolyzed per second per head, that is, a head is a single motor domain, 200 is calculated from the measured values of 7 mm s1 per 35 nm stepsize) and 1052 ATP s1 h1 (20 mm s1 per www.sciencedirect.com
19 nm) for the faster measured velocity (Chara myosin XI). Interestingly, the fastest measured actin-activated ATPase rate for N. tabacum myosin XI was 76 ATP s1 h1 [46], and for Chara myosin XI, 670 20 ATP s1 h1 [47]. Detailed enzymology for N. tabacum myosin XI is still lacking, but it will be interesting to see how it supports not only rapid velocity but also a sufficiently high DR (0.8 [46]) for its processive movement (the ability to take multiple steps along actin before dissociating). Detailed transient kinetic measurements were performed on the motor domain of Chara myosin to reveal its ATPase reaction mechanism; some interesting features were found that might support the rapid ATPase cycling time [48]. Chara myosin, that was shown to posses a 19 nm step size exhibits an extremely rapid ADP-dissociation rate (>2800 s1), fast ATP-binding rate (36 mM1 s1) and ATP-induced dissociation from actin (2200 s1), as well as very rapid ATP hydrolysis (>530 s1). However, it dwells in the strongly bound state for less than 0.82 ms, which is a very short lifespan with respect to its full ATPase cycling time of 2.5 ms calculated from the ATPase rate (kcat of 390 s1). Furthermore, its low DR of
ScienceDirect The unique enzymatic and mechanistic properties of plant myosins Arnon Henn1 and Einat Sadot2 Myosins are molecular motors that move along actin-filament tracks. Plants express two main classes of myosins, myosin VIII and myosin XI. Along with their relatively conserved sequence and functions, plant myosins have acquired some unique features. Myosin VIII has the enzymatic characteristics of a tension sensor and/or a tension generator, similar to functions found in other eukaryotes. Interestingly, class XI plant myosins have gained a novel function that consists of propelling the exceptionally rapid cytoplasmic streaming. This specific class includes the fastest known translocating molecular motors, which can reach an extremely high velocity of about 60 mm s1. However, the enzymatic properties and mechanistic basis for these remarkable manifestations are not yet fully understood. Here we review recent progress in understanding the uniqueness of plant myosins, while emphasizing the unanswered questions. Addresses 1 Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel 2 The Institute of Plant Sciences, Volcani Center, PO Box 6, Bet-Dagan 5025000, Israel Corresponding authors: Henn, Arnon ([email protected]) and Sadot, Einat ([email protected])
Current Opinion in Plant Biology 2014, 22:65–70 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´
http://dx.doi.org/10.1016/j.pbi.2014.09.006 1369-5266/# 2014 Elsevier Ltd. All rights reserved.
Introduction Live-cell imaging of plant cells reveals strikingly rapid movement of organelles and vesicles, rearrangement of the endoplasmic reticulum (ER) and constant remodeling of the tonoplast. Under certain conditions slower movement of the chloroplasts and nucleus can also be observed. This intracellular dynamic is believed to enable the immotile plant to respond quickly to abiotic and biotic stresses. The machinery underlying these vigorous movements involves actin-based molecular motors, the myosins and actin microfilaments that together drive the formation of streaming patterns in plant cells. Myosins utilize ATP to perform mechanical work such as muscle contraction and www.sciencedirect.com
the translocation of cargo along actin filaments in eukaryotic cells [1]. Across all eukaryotes there are 35 known classes of myosins [2]. In plants there are two main classes, VIII, XI [3], which are widely distributed in higher and lower plants. Plant myosins are involved in various organelles’ motility [4,5,6–14], ER remodeling [15–18] and cytoplasmic streaming [19,20,21,22]. They have been found in the plasmodesmata [23–25,26] and can be involved in targeted RNA transport [27], viral spread from cell to cell [28–31] and in a plant’s size and shape determination [6,22,32]. All known myosins share a highly conserved amino-terminal motor domain followed by a neck (also referred to as the ‘lever arm’) and the tail regions. The neck region, which contains conserved IQ repeats (as many as one to six in tandem), serves as the light chains binding domains and the tail region which displays diverse interacting domains such as cargo and membrane binding motifs [1]. The myosin actin-dependent ATPase cycle includes at least six nucleotide-linked, biochemical intermediates (Figure 1a). The cycling of myosin on actin is driven by ATP binding, hydrolysis and product release, and can be described as follows (Figure 1a from left to right): ATP binding to actomyosin causes myosin head to dissociate from actin. Detached myosin hydrolyzes ATP to ADP and Pi, which drives the repriming of the lever arm to the ‘prepower stroke’ state. This post hydrolysis state can rebind actin with a higher affinity than before the hydrolysis of ATP. Upon the rebinding of myosin-ADP-Pi state to actin conformational changes within the myosin motor domain are coupled to the release of Pi and the lever arm rotation relative to the actin-bound myosin head. This lever arm rotation transition is the force-generating step, or the ‘power-stroke’, of the myosin ATPase cycle, which is next followed by ADP release, and the formation of the myosinactin nucleotide free state. Thereafter, the catalytic cycle begins again with ATP binding. Specific nucleotide states dictate the affinity of the myosin motor domain towards actin. The strength of the myosin nucleotide binding states towards actin follows from strong to weak binding affinities as such: actomyosin > actomyosin-ADP > actomyosinADP-Pi > actomyosin-ATP. Although this ATPase cycle kinetic pathway is highly conserved among all myosins studied to date, the enzymatic properties of the catalytic motor domain display very large deviations in their kinetic and thermodynamic properties, that is, each myosin isoform displays different rate and equilibrium constants for the biochemical transitions described in Figure 1a [33,34]. These different rate and equilibrium constants give raise to different ATPase cycling-times, affinities toward actin and motility rates among myosins. They are the hallmarks Current Opinion in Plant Biology 2014, 22:65–70
66 Cell biology
Figure 1
(a)
Myosin XIChara: Very fast ADP release
“Weakly Bound”
“Strongly Bound” AM + ATP
K’H
AM ATP
KA
“Strongly Bound”
AM ADP Pi
K’Pi
M + ATP
M ATP
KH
AM + ADP
AM ADP KAD
KAPi KT
Myosin V, VIII: Rate limiting Step during ATPase cycling
M ADP Pi
“Dissociated States” (b)
KPi
KA
M ADP
KD
M + ADP
“Thermodynamic Coupling box” Low TC for Myosin V, VIII High TC for Myosin XIChara.
Second strongly bound actomyosin-ADP state
“Strongly AM ADP
K’D1
AM
Bound” ADP’
K’D2
AM + ADP
Slow isomerization Current Opinion in Plant Biology
The actomyosin ATPase cycle. (a) Schematic representation of myosin’s conserved chemomechanical energy transduction pathway is shown. The reaction pathway in the black frame is the predominant pathway for the myosin ATPase cycle as described in the text. Key features in plant myosins VIII and XIChara in relation to the ATPase reaction scheme are shown. The dashed blue frame contains the key parameters for determining thermodynamic coupling (TC) between actin and ADP affinities toward myosin. Black and blue callouts mark unique features among measured parameters of plant myosins. Blue ovals highlight extremely rapid rate constants for Chara myosin XI. Red ovals mark rate and equilibrium constants that have not been measured for any plant myosin. (b) Expansion of the minimal kinetic scheme showing the transition from actomyosin-ADP to actomyosin with additional intermediate ADP state namely actomyosin-ADP0 indicative of tension-sensor myosins.
of the diverse enzymatic behavior among the myosin family classes and isoforms. In turns, these unique features enables each myosin isoform to be enzymatically adapted to its cellular functions. Here we describe the findings related to the motor properties of the two main classes of plant myosins and discuss their enzymatic adaptation to the relevant unique cellular functions in plants.
Class VIII myosins The first plant myosin to be cloned and sequenced was Arabidopsis thaliana myosin 1 (ATM1). Phylogenetic analysis based on its motor domain placed ATM1 in a new myosin class VIII [35]. Specific antibodies against its tail domain revealed its subcellular localization in the plasmodesmata and newly formed cell plates in the roots of maize and cress (Lepidium sativum) [36]. Subsequent green fluorescent protein (GFP) fusions confirmed these observations [25,26,37], and also suggested its localization in ER junctions [25]. Analysis of the knockout plant of all five myosin VIII transcripts of P. patens revealed that this class has a role in development and hormone Current Opinion in Plant Biology 2014, 22:65–70
homeostasis [32]. Recent detailed transient kinetic and thermodynamic studies of myosin VIII have revealed its enzymatic properties [26]. It was found that ADP release (at physiological levels of Mg2+) [26] is the rate-limiting step (the slowest transition within one complete ATPaes cycle, Figure 1a) in myosin VIII’s ATPase cycle, which contributes to the long-lived and stronglybound actomyosin-ADP state during the ATPase cycle and to high duty ratio (DR — see below). The cycling time is defined as a single complete cycle of ATP binding, hydrolysis and product release (Figure 1a). Myosin VIII displays a relatively slow to moderate ATPase cycle, with a slow motility rate that is highly regulated by Mg2+ [26]. The latter characterize local tension-bearing myosins rather than fast moving transporters [33,38]. In addition, the two states, with fast and slow dissociation rates (Figure 1b), observed in myosin VIII [26] suggest that additional work can be achieved with the slower step of ADP dissociation [38]. This is a unique feature that is found in several tension-sensor myosins [33]. Furthermore, myosin VIII displays a very weak thermodynamic www.sciencedirect.com
Plant myosins Henn and Sadot 67
coupling (TC) ratio of 2.3 [26]. TC is a measure of how the affinity of myosin to actin is affected by its binding to ADP, in comparison to how its binding to ADP is affected by actin. This simply reflects the degree of communication between the nucleotide and actin binding sites, and is derived from the free energy detailed balance of the ADP states. Detailed balance requires that in the absence of external energy input or consumption the product of the four equilibrium constants equal unity and, therefore, K 0D =K D ¼ K A =K AD (Figure 1a). Tensionsensor myosins have been shown to display very weak TC or a low ratio close to unity between the equilibrium constants [33,38]. A weak TC permits strong binding to actin in the presence of ADP, and hence to bear tension in the strongly bound ADP states. Lastly, the kinetic adaptation of plant myosin VIII for its function also stems from its measured high DR of 0.9 [26]. The DR is defined as the fraction of time that myosin spends strongly bound to actin during the complete ATPase cycle. Thus, the detailed enzymological studies of myosin VIII strongly suggest that it is enzymatically adapted to maintaining tension, such as that required for ER tethering to the plasma membrane for proper plasmodesmatal function [39]. However, the latter assumption requires in-vivo validations.
Class XI myosins Class XI myosins from Chara corallina are the fastest actin-based motors characterized to date, even faster than myosin XI members in higher plants. Class XI myosins show sequence and structural similarities to vertebrates’ transporter class V myosins [40]. Both contain a long lever arm composed of six IQ motifs, indicative of a large step size, and a coiled-coil motif, which favors a doubleheaded (dimer) myosins’ oligomeric state [41]. Despite these similarities to myosin class V, the velocity measurements by in vitro motility assay for plant myosins have demonstrated remarkably higher rates. For purified C. corallina myosin XI, the measured velocities range from 20 mm s1 up to 60 mm s1 [42–44], and for myosin XI from Nicotiana tabacum and Arabidopsis this is about 7 mm s1. These measurements are up to 150-folds and 20-folds respectively, faster than myosin V [45]. Purified myosin XI from N. tabacum was found to have a step size of 35 nm [46]. This step size was similar to that measured for myosin V, which has an identical lever arm length of six IQ motifs [45]. The step size measured for the faster Chara myosin XI was 19 nm [44]. The extraordinarily high velocity of myosin XI does not result from an unusually large step size. Calculations combining velocity with step size suggested that the ATPase cycling time for the slower measured velocity (myosin XI from N. tabacum and Arabidopsis) should be 200 ATP s1 h1 (s1 h1 is define as moles of ATP hydrolyzed per second per head, that is, a head is a single motor domain, 200 is calculated from the measured values of 7 mm s1 per 35 nm stepsize) and 1052 ATP s1 h1 (20 mm s1 per www.sciencedirect.com
19 nm) for the faster measured velocity (Chara myosin XI). Interestingly, the fastest measured actin-activated ATPase rate for N. tabacum myosin XI was 76 ATP s1 h1 [46], and for Chara myosin XI, 670 20 ATP s1 h1 [47]. Detailed enzymology for N. tabacum myosin XI is still lacking, but it will be interesting to see how it supports not only rapid velocity but also a sufficiently high DR (0.8 [46]) for its processive movement (the ability to take multiple steps along actin before dissociating). Detailed transient kinetic measurements were performed on the motor domain of Chara myosin to reveal its ATPase reaction mechanism; some interesting features were found that might support the rapid ATPase cycling time [48]. Chara myosin, that was shown to posses a 19 nm step size exhibits an extremely rapid ADP-dissociation rate (>2800 s1), fast ATP-binding rate (36 mM1 s1) and ATP-induced dissociation from actin (2200 s1), as well as very rapid ATP hydrolysis (>530 s1). However, it dwells in the strongly bound state for less than 0.82 ms, which is a very short lifespan with respect to its full ATPase cycling time of 2.5 ms calculated from the ATPase rate (kcat of 390 s1). Furthermore, its low DR of
