Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic (original) (raw)
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ADP Inhibition of Myosin V ATPase Activity
Biophysical Journal, 2000
The kinetic mechanism of myosin V is of great interest because recent evidence indicates that the two-headed myosin V molecule functions as a processive motor, i.e., myosin V is capable of moving along an actin filament for many catalytic cycles of the motor without dissociating. Three recent publications assessing the kinetics of single-headed myosin V provide different conclusions regarding the mechanism, particularly the rate-limiting step of the cycle. One study (De La Cruz et al., 1999, Proc. Natl. Acad. Sci. USA. 96:13726 -13731) identifies ADP release as the rate-limiting step and provides a kinetic explanation for myosin V processivity. The others (Trybus et al., 1999, J. Biol. Chem. 274:27448 -27456; Wang et al., 2000, J. Biol. Chem. 275:4329 -4335) do not identify the rate-limiting step but conclude that it is not ADP release. We show experimental and simulated data demonstrating that the inconsistencies in the reports may be due to difficulties in the measurement of the steady-state ATPase rate. Under standard assay conditions, ADP competes with ATP, resulting in product inhibition of the ATPase rate. This presents technical problems in analyzing and interpreting the kinetics of myosin V and likely of other members of the myosin family with high ADP affinities.
JCB Article Identification of an organelle-specific myosin V receptor
2013
Class V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the
Mechanism of action of myosin X, a membrane-associated molecular motor
We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steadystate ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin⅐ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.
Identification of an organelle-specific myosin V receptor
The Journal of Cell Biology, 2003
lass V myosins are widely distributed among diverse organisms and move cargo along actin filaments. Some myosin Vs move multiple types of cargo, where the timing of movement and the destinations of selected cargoes are unique. Here, we report the discovery of an organelle-specific myosin V receptor. Vac17p, a novel protein, is a component of the vacuole-specific receptor for Myo2p, a Saccharomyces cerevisiae myosin V. Vac17p interacts with the Myo2p cargo-binding domain, but not with vacuole inheritance-defective myo2 mutants that have single amino acid changes within this region. Moreover, a region of the C Myo2p tail required specifically for secretory vesicle transport is neither required for vacuole inheritance nor for Vac17p-Myo2p interactions. Vac17p is localized on the vacuole membrane, and vacuole-associated Myo2p increases in proportion with an increase in Vac17p. Furthermore, Vac17p is not required for movement of other cargo moved by Myo2p. These findings demonstrate that Vac17p is a component of a vacuole-specific receptor for Myo2p. Organelle-specific receptors such as Vac17p provide a mechanism whereby a single type of myosin V can move diverse cargoes to distinct destinations at different times.
Myosin V: regulation by calcium, calmodulin, and the tail domain
The Journal of Cell Biology, 2004
alcium activates the ATPase activity of tissue-purified myosin V, but not that of shorter expressed constructs. Here, we resolve this discrepancy by comparing an expressed full-length myosin V (dFull) to three shorter constructs. Only dFull has low ATPase activity in EGTA, and significantly higher activity in calcium. Based on hydrodynamic data and electron microscopic images, the inhibited state is due to a compact conformation that is possible only with the whole molecule. The paradoxical finding that dFull moved actin in EGTA suggests that binding C of the molecule to the substratum turns it on, perhaps mimicking cargo activation. Calcium slows, but does not stop the rate of actin movement if excess calmodulin (CaM) is present. Without excess CaM, calcium binding to the high affinity sites dissociates CaM and stops motility. We propose that a folded-to-extended conformational change that is controlled by calcium and CaM, and probably by cargo binding itself, regulates myosin V's ability to transport cargo in the cell.
Biochemistry, 2003
Much interest has centered on two surface loops in the motor domain to explain the differences in enzymatic and mechanical properties of myosin isoforms. We showed that two invariant lysines at the C-terminal end of loop 2, which is part of the actin-binding interface, are required to obtain actin activation [Joel et al. (2001) J. Biol. Chem. 276, 2998-3003]. Here we investigate the effects of increasing positive charge in the variable portion of loop 2 of smooth muscle heavy meromyosin (smHMM). Increasing the net positive charge by +4 increased the affinity for actin in the presence and absence of ATP. The K m for actin-activated ATPase activity decreased 15-fold, but V max was unchanged, showing that "weak binding" of myosin for actin can be significantly strengthened without increasing the rate-limiting step for V max. The mutant HMM had slower rates of in vitro motility and ADP release compared to WT HMM. ADP release and motility, which were both salt-dependent, correlated linearly with each other. Loop 2 thus plays a major role in setting the affinity for actin but also affects ADP release and motility. Because the actin-and nucleotide-binding regions communicate, mutations to one region can impact multiple facets of myosin's mechanical and enzymatic properties.
Functional Characterization of Human Myosin-18A and its Interaction Partners
Biophysical Journal, 2012
Developmental and electrophysiological studies have shown that the cochlear function is altered in these mice. We carried out a detailed kinetic analysis of the baculoviral expressed truncated shaker-1 construct to assess whether the ATP hydrolysis mechanism is altered in these mice. The basal steadystate rate (0.03 s À1) is activated by actin only 1.5-fold. ATP binding is relatively fast (1.22 uM À1 s À1) and the dissociation of the myosin from the actomyosin complex is the same as for the wild type myosin VIIa (400 s À1). ADP binding to (1.8 uM À1 s À1) and the dissociation (1.5 s À1) from the actomyosin complex are also similar to the wild type. Phosphate release from the M-ADP-P i complex is slow (0.07 s À1) and is not activated by actin. Quenched flow experiments show that there is no Pi burst and the rate of the hydrolysis is 0.12 s À1. The R502 residue is in a small loop that is in the middle of an a-helical region which aligns with the switch-2 helix and is the continuation of the relayloop. The sequence in the loop is changed from NRPM to NPPM which is likely to alter the conformation of the helix-loop-helix structure that is relatively straight in all myosins. Consequently the rate constants of the steps leading into the power stroke (hydrolysis and phosphate dissociation) are reduced greater than 10-fold whereas the post-power stoke steps are essentially unchanged. As a result the shaker-1 mutant protein has a severely compromised ATP hydrolysis mechanism in which < 10% of the normal fraction of the force-producing crossbridges are formed.