Inter-dependence of dimerization and organelle binding in myosin XI

Cytoplasmic streaming is a ubiquitous process in plant cells that is thought to be driven by the active movement of myosin XI motor proteins along actin filaments. These myosin motors bind to organelles through their C-terminal globular tail domain, although recent studies have also suggested a role for the central coiled-coil region during organelle binding. Here we have investigated the relationship between these two protein domains of MYA1, an Arabidopsis myosin XI, in a series of in vivo experiments demonstrating that dimerization of the coiled-coil region stabilizes organelle binding of the globular tail. Surprisingly, yeast two-hybrid assays, bimolecular fluorescence complementation, Förster resonance energy transfer and in vitro pull-down experiments all demonstrated that dimerization of the 174-residue MYA1 coiled coils by themselves was unstable. Furthermore, only the first of the two major coiled-coil segments in MYA1 contributed significantly to dimer formation. Interestingly, dimerization of myosin tail constructs that included the organelle-binding globular tail was stable, although the globular tails by themselves did not interact. This suggests an inter-dependent relationship between dimerization and organelle binding in myosin XI, whereby each process synergistically stimulates the other.     

The tail that wags the dog: the globular tail domain defines the function of myosin V/XI

Actin-based organelle movements are driven by the related multifunctional myosin motors of class V in animals and fungi and class XI in plants. The versatility of these motors depends critically on their C-terminal globular tail domain that allows them to bind to a broad variety of cargo molecules. Regulation of this motor-cargo attachment is frequently employed to modulate organelle movement. While the overall structure of the cargo-binding globular tail appears to be conserved between myosin V and XI, it has become apparent that the motor-cargo interactions differ widely even within a single organism and involve protein complexes with different architecture and completely unrelated protein domains. At the same time, indirect evidence suggests that adaptor or receptor dimerization could facilitate efficient myosin capture. Comparison of myosin V and XI across the large evolutionary distance between animals and plants will likely reveal more fundamental insights into these important motors.     

Myosin V attachment to cargo requires the tight association of two functional subdomains

The myosin V carboxyl-terminal globular tail domain is essential for the attachment of myosin V to all known cargoes. Previously, the globular tail was viewed as a single, functional entity. Here, we show that the globular tail of the yeast myosin Va homologue, Myo2p, contains two structural subdomains that have distinct functions, namely, vacuole-specific and secretory vesicle-specific movement. Biochemical and genetic analyses demonstrate that subdomain I tightly associates with subdomain II, and that the interaction does not require additional proteins. Importantly, although neither subdomain alone is functional, simultaneous expression of the separate subdomains produces a functional complex in vivo. Our results suggest a model whereby intramolecular interactions between the globular tail subdomains help to coordinate the transport of multiple distinct cargoes by myosin V.     

A point mutation in the cargo-binding domain of myosin V affects its interaction with multiple cargoes

Class V myosins move diverse intracellular cargoes, which attach via interaction of cargo-specific proteins to the myosin V globular tail. The globular tail of the yeast myosin V, Myo2p, contains two structural and functional subdomains. Subdomain I binds to the vacuole-specific protein, Vac17p, while subdomain II likely binds to an as yet unidentified secretory vesicle-specific protein. All functions of Myo2p require the tight association of subdomains I and II, which suggests that binding of a cargo to one subdomain may inhibit cargo-binding to a second subdomain. Thus, two types of mutations are predicted to specifically affect a subset of Myo2p cargoes: first are mutations within a cargo-specific binding region; second are mutations that mimic the inhibited conformation of one of the subdomains. Here we analyze a point mutation in subdomain I, myo2-2(G1248D), which is likely to be this latter type of mutation. myo2-2 has no effect on secretory vesicle movement. The secretory vesicle binding site is in subdomain II. However, myo2-2 is impaired in several Myo2p-related functions. While subdomains I and II of myo2-2p tightly associate, there are measurable differences in the conformation of its globular tail. Based solely on the ability to restore vacuole inheritance, a set of intragenic suppressors of myo2-2 were identified. All suppressor mutations reside in subdomain I. Moreover, subdomain I and II interactions occurred in all suppressors, demonstrating the importance of subdomain I and II association for Myo2p function. Furthermore, 3 of the 10 suppressors globally restored all tested defects in myo2-2. This large proportion of global suppressors strongly suggests that myo2-2(G1248) causes a conformational change in subdomain I that simultaneously affects multiple cargoes.     

Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin

Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.     

Emergent complexity in Myosin V-based organelle inheritance

How is adaptability generated in a system composed of interacting cellular machineries, each with a separate and functionally critical job to perform? The machinery for organelle inheritance is precisely one such system, requiring coordination between robust and ancient cellular modules, including the cell cycle, cytoskeleton, and organelle biogenesis/identity. Budding yeasts have emerged as powerful models to study these processes, which are critical for cellular survival, propagation, and differentiation, as organelles must compete for access to myosin V motors that travel along polarized actin cables to vectorially deliver bound cargo to the bud. Under the direction of the cell cycle, myosin V motors are recruited to organelles by specific interactions between their carboxyl-terminal globular tail domains and organelle-specific receptors. We used comparative genomics, phylogenetics, and secondary structure modeling to characterize the evolutionary history of these organelle-specific receptors. We find that while some receptors are retained widely across the animals and fungi, others are limited primarily to the Saccharomycetaceae family of budding yeast, with the emergent pattern of a conserved biogenic and inheritance factor often paired with an evolutionarily novel inheritance adaptor. We propose an evolutionary model whereby the emergence of myosin V-based organelle inheritance has utilized mechanisms of paralogy, mutation, and the appearance of pliable evolutionarily novel adaptor proteins. Our findings suggest an overarching evolutionary mechanism for how diverse cargoes compete for a single myosin V motor in organelle transport and detail one system's solution to obtaining evolutionary adaptability amongst constrained cellular modules.     

Cloning and characterization of a novel RING finger protein that interacts with class V myosins.

We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V. Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles. BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain. A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb). This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene. Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells. We also found that the RBCC domain of BERP is involved in protein dimerization. Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor. Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport.     

Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog

The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.     

Structural basis for myosin V discrimination between distinct cargoes

Myosin V molecular motors move cargoes on actin filaments. A myosin V may move multiple cargoes to distinct places at different times. The cargoes attach to the globular tail of myosin V via cargo-specific receptors. Here we report the crystal structure at 2.2 A of the myosin V globular tail. The overall tertiary structure has not been previously observed. There are several patches of highly conserved regions distributed on the surface of the tail. These are candidate attachment sites for cargo-specific receptors. Indeed, we identified a region of five conserved surface residues that are solely required for vacuole inheritance. Likewise, we identified a region of five conserved surface residues that are required for secretory vesicle movement, but not vacuole movement. These two regions are at opposite ends of the oblong-shaped cargo-binding domain, and moreover are offset by 180 degrees. The fact that the cargo-binding areas are distant from each other and simultaneously exposed on the surface of the globular tail suggests that major targets for the regulation of cargo attachment are organelle-specific myosin V receptors.     

Ypt32p and Mlc1p bind within the vesicle binding region of the class V myosin Myo2p globular tail domain

Myosin V is an actin-based motor essential for a variety of cellular processes including skin pigmentation, cell separation and synaptic transmission. Myosin V transports organelles, vesicles and mRNA by binding, directly or indirectly, to cargo-bound receptors via its C-terminal globular tail domain (GTD). We have used the budding yeast myosin V Myo2p to shed light on the mechanism of how Myo2p interacts with post-Golgi carriers. We show that the Rab/Ypt protein Ypt32p, which associates with membranes of the trans -Golgi network, secretory vesicles and endosomes and is related to the mammalian Rab11, interacts with the Myo2p GTD within a region previously identified as the 'vesicle binding region'. Furthermore, we show that the essential myosin light chain 1 (Mlc1p), required for vesicle delivery at the mother-bud neck during cytokinesis, binds to the Myo2p GTD in a region overlapping that of Ypt32p. Our data are consistent with a role of Ypt32p and Mlc1p in regulating the interaction of post-Golgi carriers with Myo2p subdomain II.     

Activation of myosin Va function by melanophilin, a specific docking partner of myosin Va

It is known that melanophilin is a myosin Va-targeting molecule that links myosin Va and the cargo vesicles in cells. Here we found that melanophilin directly activates the actin-activated ATPase activity of myosin Va and thus its motor activity. The actin-activated ATPase activity of the melanocyte-type myosin Va having exon-F was significantly activated by melanophilin by 4-fold. Although Rab27a binds to myosin Va/melanophilin complex, it did not affect the melanophilin-induced activation of myosin Va. Deletion of the C-terminal actin binding domain and N-terminal Rab binding domain of melanophilin resulted in no change in the activation of the ATPase by melanophilin, indicating that the myosin Va binding domain (MBD) is sufficient for the activation of myosin Va. Among MBDs, the interaction of MBD-2 with exon-F of myosin Va is critical for the binding of myosin Va and melanophilin, whereas MBD-1 interacting with the globular tail of myosin Va plays a more significant role in the activation of myosin Va ATPase activity. This is the first demonstration that the binding of the cargo molecule directly activates myosin motor activity. The present finding raises the idea that myosin motors are switched upon their binding to the cargo molecules, thus avoiding the waste of ATP consumption.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region

Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.     

Arabidopsis myosin XIK interacts with the exocyst complex to facilitate vesicle tethering during exocytosis

Myosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to several exocyst subunits in vitro and functional fluorescently tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the rate of appearance and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes with high spatiotemporal resolution imaging and pair-wise colocalization of myosin XIK, exocyst subunits, and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides insights about the dynamic regulation of exocytosis in plants.     

Head-to-tail regulation is critical for the in vivo function of myosin V

Cell organization requires regulated cargo transport along cytoskeletal elements. Myosin V motors are among the most conserved organelle motors and have been well characterized in both yeast and mammalian systems. Biochemical data for mammalian myosin V suggest that a head-to-tail autoinhibitory interaction is a primary means of regulation, but the in vivo significance of this interaction has not been studied. Here we generated and characterized mutations in the yeast myosin V Myo2p to reveal that it is regulated by a head-to-tail interaction and that loss of regulation renders the myosin V constitutively active. We show that an unregulated motor is very deleterious for growth, resulting in severe defects in Myo2-mediated transport processes, including secretory vesicle transport, mitochondrial inheritance, and nuclear orientation. All of the defects associated with motor misregulation could be rescued by artificially restoring regulation. Thus, spatial and temporal regulation of myosin V in vivo by a head-to-tail interaction is critical for the normal delivery functions of the motor.     

Walking to work: roles for class V myosins as cargo transporters

Cells use molecular motors, such as myosins, to move, position and segregate their organelles. Class V myosins possess biochemical and structural properties that should make them ideal actin-based cargo transporters. Indeed, studies show that class V myosins function as cargo transporters in yeast, moving a range of organelles, such as the vacuole, peroxisomes and secretory vesicles. There is also increasing evidence in vertebrate cells that class V myosins not only tether organelles to actin but also can serve as short-range, point-to-point organelle transporters, usually following long-range, microtubule-dependent organelle transport.     

Extensibility of the Extended Tail Domain of Processive and Nonprocessive Myosin V Molecules

Myosin V is a single-molecule motor that moves organelles along actin. When myosin V pulls loads inside the cell in a highly viscous environment, the force on the motor is unlikely to be constant. We propose that the tether between the single-molecule motor and the cargo (i.e., the extended tail domain of the molecule) must be able to absorb the sudden mechanical motions of the motor and allow smooth relaxation of the motion of the cargo to a new position. To test this hypothesis, we compared the elastic properties of the extended tail domains of processive (mouse myosin Va) and nonprocessive (Drosophila myosin V) molecular motors. The extended tail domain of these myosins consists of mechanically strong coiled-coil regions interspersed with flexible loops. In this work we explored the mechanical properties of coiled-coil regions using atomic force microscopy. We found that the processive and nonprocessive coiled-coil fragments display different unfolding patterns. The unfolding of coiled-coil structures occurs much later during the atomic force microscopy stretch cycle for processive myosin Va than for nonprocessive Drosophila myosin V, suggesting that this elastic tether between the cargo and motor may play an important role in sustaining the processive motions of this single-molecule motor.     

Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

Background  

Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2), which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins.     

Myosin V plays an essential role in the thyroid hormone-dependent endocytosis of type II iodothyronine 5'-deiodinase

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.     

Regulated phosphorylation of budding yeast's essential myosin V heavy chain, Myo2p

The tail of the yeast myosin V encoded by Myo2p is known to bind several receptors for cargo delivery along polarized actin cables. However, it is not known how Myo2p activity is regulated or how it selects between cargoes. Here we show that Myo2p is reversibly phosphorylated in vivo. A short peptide at the N-terminal end of the cargo-binding domain contains three residues contributing to single or doubly phosphorylated species. We confirm that the tail consists of two proteolytically resistant subdomains and identify a functionally important region N-terminal to subdomain 1 that includes the phosphorylation sites. Mutagenesis of the phosphorylation sites to alanine abolished a mobility shift diagnostic of phosphorylation, whereas mutagenesis to glutamic acid produced the shift and the formation of an additional phosphorylated species. These substitutions did not affect overall cell growth. However, one of the sites is predicted to be a substrate of cAMP-dependent protein kinase (PKA), and yeast expressing Myo2p with alanine substitutions is resistant to otherwise lethal overexpression of PKA, whereas the glutamic acid mutant is supersensitive to overexpression of PKA. These results suggest that in yeast, Myo2p is subject to phosphoregulation involving a PKA-related signaling pathway.