Organelle Targeting of Myosin XI Is Mediated by Two Globular Tail Subdomains with Separate Cargo Binding Sites

Abstract

Myosin XI are actin-based molecular motors that are thought to drive organelle movements in plants, analogous to myosin V in animals and fungi. Similar domain structure of these myosins suggests that binding to organelles may occur via the globular tail domain in both types of motors, even though sequence similarity is low. To address this hypothesis, we developed a structure homology model for the globular tail of MYA1, a myosin XI from Arabidopsis, based on the known structure of yeast myosin V (Myo2p) globular tail. This model suggested an interaction between two subdomains of the globular tail which was verified by yeast two-hybrid assay and by in vivo bimolecular fluorescence complementation (BiFC). Interface mapping demonstrated that this subdomain interaction depends critically on the C terminus of helix H6 as well as three specific residues in helices H3 and H15, consistent with the structural prediction. The reconstituted globular tails of several Arabidopsis myosin XIs in BiFC assays targeted to peroxisomes in plant cells, identifying this domain as sufficient for cargo binding. Unlike myosin V, either subdomain of myosin XI alone was targeting-competent and responsible for association with different organelles. In addition, our data suggest that organelle binding is regulated by an allosteric interaction between two tail subdomains. We conclude that the globular tail of myosin XI shares a similar structure with that of myosin V, but has evolved plant-specific cargo binding mechanisms.Myosins are efficient molecular motor proteins which convert chemical energy from ATP hydrolysis to physical force to move along cytoskeletal actin filaments (1). Based on sequence comparison of myosin motor domains, these nearly ubiquitous eukaryotic motors can be grouped into 24 subfamilies designated as class I to XXIV (2). Plants from green algae to flowering plants encode only two classes, myosin VIII and XI (3). A number of myosin XI isoforms have been identified from a variety of organisms by biochemical isolation (4, 5), molecular cloning (6 -8), or genome-wide sequence analysis (9, 10). Little is known about the specific cellular function of class XI myosins in plants, but it is generally assumed that they are responsible for force generation for organelle transport during cytoplasmic streaming (11).This assumption is based on three lines of evidence. First, disruption of organelle movements in plant cells by anti-actin drug or myosin ATPase inhibitor treatment strongly indicated that organelle motility depends on both actin and myosin (12-16). Second, myosin XI was found to associate with a diverse array of organelles in plant cells by immunostaining (17-20), YFP labeling (21), and co-purification studies (22). Finally, there is an overall structural similarity between class XI and class V myosins, the latter being responsible for organelle movement in fungi and animals (23). In particular: (i) both classes have the same domain arrangement, including a catalytic motor in the head, six copies of calmodulin bindin...

Myosin XI-K Is Required for Rapid Trafficking of Golgi Stacks, Peroxisomes, and Mitochondria in Leaf Cells of Nicotiana benthamiana

“…However, it appears that Arabidopsis myosin XI-2 is (almost) as significant as myosin XI-K for movement of these three organelles in Arabidopsis, but not in N. benthamiana, where the myosin XI-2 roles appear to be subordinate (Peremyslov et al, 2008). These findings are compatible with the reports that Arabidopsis myosin XI-2 associates with peroxisomes (Hashimoto et al, 2005;Li and Nebenfuhr, 2007;Reisen and Hanson, 2007).…”

“…The angiosperms have two classes of myosins, VIII and XI, with the latter being the largest. Plant class XI myosins share a close relationship with the class V myosins found in metazoa and fungi (Foth et al, 2006;Li and Nebenfuhr, 2007). Class V myosins are processive molecular motors that function in organelle and vesicle transport and partitioning during cell division, mitotic spindle positioning, localization of mRNA, and establishment of cell polarity (Pruyne et al, 2004;Sellers and Veigel, 2006;Desnos et al, 2007).…”

“…However, because two independent studies, including an accompanying article (Peremyslov et al, 2008), failed to reproduce the latter phenotype (Hashimoto et al, 2005), its significance remains unclear. Recent publications demonstrated that GTDs of class XI myosins are structurally and functionally similar to GTDs of yeast class V myosins and are involved in binding the various organelles as their cargoes (Li and Nebenfuhr, 2007;Reisen and Hanson, 2007). In addition, Arabidopsis myosin XI-K was implicated in the root hair growth (Ojangu et al, 2007).…”

PTC1Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

“…Rab27a, through geranylgeranylation, attaches to the melanosome membrane, and melanophilin connects myosin-Va and Rab27a. Myosin-V-based intracellular movement has been analyzed in many other eukaryotes, including frogs, fish, mammals, and plants; plant myosin-XI is the functional homologue of yeast and vertebrate myosin-V ( Kinkema and Schiefelbein, 1994;Provance and Mercer, 1999;Tuma and Gelfand, 1999;Desnos et al, 2007;Li and Nebenfuhr, 2007;Sheets et al, 2007;Shimmen, 2007).…”

“…Rab27a, through geranylgeranylation, attaches to the melanosome membrane, and melanophilin connects myosin-Va and Rab27a. Myosin-V-based intracellular movement has been analyzed in many other eukaryotes, including frogs, fish, mammals, and plants; plant myosin-XI is the functional homologue of yeast and vertebrate myosin-V ( Kinkema and Schiefelbein, 1994;Provance and Mercer, 1999;Tuma and Gelfand, 1999;Desnos et al, 2007;Li and Nebenfuhr, 2007;Sheets et al, 2007;Shimmen, 2007).Genetic screens have identified several molecules that play a role in organelle inheritance. For example, vacuole inheritance utilizes the actin cytoskeleton and the class-V myosin complex, composed of Myo2p, Vac17p, and Vac8p (Hill et al, 1996;Wang et al, 1998;Ishikawa et al, 2003;Tang et al, 2003).…”