Roles and regulation of myosin V interaction with cargo

Abstract

A major question in cell biology is, how are organelles and large macromolecular complexes transported within a cell? Myosin V molecular motors play critical roles in the distribution of organelles, vesicles, and mRNA. Mis-localization of organelles that depend on myosin V motors underlie diseases in the skin, gut, and brain. Thus, the delivery of organelles to their proper destination is important for animal physiology and cellular function. Cargoes attach to myosin V motors via cargo specific adaptor proteins, which transiently bridge motors to their cargoes. Regulation of these adaptor proteins play key roles in the regulation of cargo transport. Emerging studies reveal that cargo adaptors play additional essential roles in the activation of myosin V, and the regulation of actin filaments. Here, we review how motor-adaptor interactions are controlled to regulate the proper loading and unloading of cargoes, as well as roles of adaptor proteins in the regulation of myosin V activity and the dynamics of actin filaments.

Introduction

The correct positioning of contents within a cell is crucial to cellular function. The movement and delivery of cellular materials, such as organelles, is essential for response to stimuli and for the establishment and maintenance of homeostasis and cellular identity. Organelles are dynamic and change morphology and location depending on cellular requirements. For example, during mitosis in mammalian cell lines, the contents of the mother cell are distributed between the two daughter cells (Jongsma et al., 2015). Similarly, during the cell-cycle of budding yeast, portions of each organelle are targeted from the mother to the bud (Knoblach and Rachubinski, 2015; Pruyne et al., 2004; Weisman, 2006). Organelle transport is also critical in non-dividing cells. One example are melanosomes, lysosome-like pigment containing organelles, which are trafficked to the dendritic tips of post mitotic melanocytes (Barral and Seabra, 2004). Similarly, organelles are targeted to axons and dendrites in post mitotic neurons (Maeder et al., 2014). Organelle transport frequently occurs in response to changes in the cellular environment. For example, lysosomes change localization in response to starvation. This movement is correlated with the degradation of the lysosomal contents that were loaded from other parts of the cell (Oyarzun et al., 2019). This phenomenon is likely linked to prior observations that the luminal pH of lysosomes correlates with whether they are at the cell periphery or located to a perinuclear region (Johnson et al., 2016). Along similar lines, in neurons, endosomes travel from distal regions of the axon to the soma and mature into degradative lysosomes (Winckler et al., 2018).

Transport of many organelles and macromolecular complexes is mediated by molecular motors. In many eukaryotic cells, including mammalian cells, long range transport occurs via kinesin and dynein on microtubules (Cross and Dodding, 2019; Hoogenraad and Akhmanova, 2016; Reck-Peterson et al., 2018). At the cell periphery, some cargoes are then transferred from kinesin to myosin V motors for short range transport and/or tethering on actin networks (Hancock, 2014). In some cases, proper cargo positioning requires competition between a kinesin and myosin V motor (Lu et al., 2020). In addition, myosin V motors may also function in long-range movement independent of kinesins via the coordination of myosin V with dynamic actin filaments (Evans et al., 2014).

Myosin V motors are dimers and move via hand-over-hand movement of the motor domains. This movement is powered by ATP hydrolysis, which induces conformational changes in the motor to alternate between tight and relaxed binding to actin filaments (reviewed in (Hammer and Sellers, 2012; Heissler et al., 2017; Sweeney and Houdusse, 2004; Trybus, 2008)).

Accurate transport by myosin V motors requires several regulated steps (Figure 1): 1) activation of the motor, which involves its release from an autoinhibited state, 2) attachment of cargos which involves regulation of both the cargo binding domain of the motor and cargo-specific adaptor proteins, 3) dynamic assembly of the actin filaments by selected cargo adaptors, 4) disassembly of actin filaments at the cargo destination, 5) detachment of the cargo from the motor, and 6) return of the motor to an autoinhibited state. Here, we review current knowledge of how actin-based myosin V motors and cargo-specific adaptor proteins interact to ensure proper cargo delivery. Moreover, we discuss the roles of cargo adaptors in myosin V activation, and remodeling of the local actin cytoskeleton.

Figure 1. Schematic of a proposed pathway for myosin V-dependent delivery of cargoes.

Delivery of myosin V-dependent cargoes requires at least six steps: 1) activation of the motor, which involves its release from an autoinhibited state, 2) attachment of cargos which involves regulation of both the cargo binding domain of the motor as well as cargo adaptor proteins, 3) dynamic assembly of the actin filaments by selected cargo adaptors, 4) disassembly of actin filaments at the cargo destination, 5) detachment of the cargo from the motor, and 6) return of the motor to an autoinhibited state.

Mechanisms for myosin V selection of cargoes

Most myosin V motors carry multiple types of cargoes to distinct places at different times. This is achieved via mechanisms that control when and where a specific cargo will attach to the motor. The architecture of the cargo-binding domain of myosin V contributes to this regulation. In addition, the majority of the regulation is due to cargo-specific adaptor proteins. Moreover, post-translational modification of the cargo-binding domains, as well as the adaptor proteins, likely play key roles.

The Myo2 cargo-binding domain

The globular tail domain, also called the cargo binding domain, of the yeast myosin V, Myo2, resides in a region of approximately 500 amino acids at the C-terminus. Mammalian myosin Va, Vb, and Vc have a similar region. Early indications that the globular tail functions as a cargo binding domain came from studies of yeast Myo2 showing that specific point mutations in this domain abolish the ability of Myo2 to move the vacuole (Catlett et al., 2000; Catlett and Weisman, 1998). These findings were further supported by studies of myosin Va, which showed that this region binds melanophilin, and that a tripartite complex with Rab27a was important for transport of melanocytes (Fukuda and Kuroda, 2002; Hume et al., 2002; Nagashima et al., 2002; Provance et al., 2002; Wu et al., 2002). High-resolution structures of the cargo-binding domains of yeast Myo2 (Pashkova et al., 2006) and mammalian myosin Va, Vb, and Vc (Nascimento et al., 2013; Pylypenko et al., 2013; Velvarska and Niessing, 2013), indicated that the cargo binding domains of myosin Va, Vb, Vc, and yeast Myo2 are highly similar.

Transport of cargoes occurs via myosin V attachment to cargo specific adaptor proteins. Each Myo2-dependent cargo has a unique cargo-specific adaptor, which physically bridges the specific cargo to the motor (Altmann et al., 2008; Arai et al., 2008; Catlett et al., 2000; Catlett and Weisman, 1998; Chernyakov et al., 2013; Eves et al., 2012; Fagarasanu et al., 2006; Fortsch et al., 2011; Hoepfner et al., 2001; Itoh et al., 2002; Jin et al., 2011; Lipatova et al., 2008; Otzen et al., 2012; Pashkova et al., 2006; Reck-Peterson et al., 1999; Santiago-Tirado et al., 2011; Schott et al., 1999; Wagner et al., 2002; Yin et al., 2000). In some cases, a single cargo requires two adaptors that bind to distinct regions of the Myo2 cargo-binding domain (Arai et al., 2008; Chernyakov et al., 2013). Importantly, the seven cargoes tested bind to just one of three spatially distinct areas on the Myo2 globular tail that participate in cargo binding (Eves et al., 2012). Moreover, due to potential connections via shared loops, these areas may communicate with each other through potential structural changes during cargo binding. Indeed, recent structures of Myo2 bound to peptides of cargo adaptor proteins, provide further indication that significant structural changes occur (Tang et al., 2019). A caveat is that a subset of these structures were obtained by artificially tethering the peptide to Myo2, which may have induced a conformation change or binding site that does not occur in cells. Similarly, cargo binding sites on myosin Va overlap, and importantly, myosin Va also undergoes conformational changes induced via binding to specific cargo adaptors (Pylypenko et al., 2013; Wei et al., 2013).

The overlap of cargo-binding domains on myosin V likely plays a regulatory role. For yeast Myo2, at least four cargoes bind to overlapping sites in an area, which includes a Rab GTPase binding site (Eves et al., 2012). Analysis of a simpler area on Myo2, with overlap of just two cargoes - the vacuole/lysosome and mitochondria, revealed that these organelles compete with each other for access to Myo2 (Eves et al., 2012). Importantly, this competition controls the volume of mitochondria and vacuoles that are distributed from the mother cell to the bud.

Cargo-specific adaptor proteins control the attachment of cargo to Myo2

Many of the cargo adaptor proteins for myosin V are not readily identified by their primary sequence or presence of specific motifs. Moreover, the adaptors are not evolutionarily conserved (Mast et al., 2012). An exception are some Rab GTPases, which are involved in cargo binding of some cargoes for both yeast Myo2 and mammalian myosin Va, Vb, and Vc. The yeast Rab GTPases Ypt31/32 (Lipatova et al., 2008), which are important for vesicle formation, link newly forming post-Golgi vesicles to Myo2. This is followed by a hand-off to the Rab GTPase Sec4 (Jin et al., 2011), which is critical for vesicle fusion with the plasma membrane. This Rab GTPase cascade promotes Myo2 attachment and detachment from secretory vesicles (Jin et al., 2011; Lipatova et al., 2008; Santiago-Tirado et al., 2011), and links secretory vesicle arrival at the plasma membrane with fusion (Donovan and Bretscher, 2012).

Notably, these Rab GTPases are not sufficient for attachment of Myo2 to secretory vesicles. Sec15, a subunit of the exocyst tethering complex, which is required for vesicle tethering to the plasma membrane, binds on the opposite side of Myo2 (Jin et al., 2011), and functions with Ypt31/32 and Sec4 in the Myo2-dependent transport of secretory vesicles. Moreover, a purified, recombinant globular tail of mammalian Va pulls down the exocyst complex from mammalian cell lysates, and interacts directly with recombinant mammalian Sec15. Furthermore, the Sec15-binding site is partially conserved between Myo2 and myosin Va, Vb, and Vc, which suggests that this is a conserved interaction (Jin et al., 2011). The significance of the Sec15 site is controversial. The same site is critical for formation of an autoinhibited state, and it has been proposed that this is the sole role of this site (Donovan and Bretscher, 2015).

Simultaneous interaction of two adaptors with the globular tail is also observed for the transport of other Myo2 cargoes. The Rab GTPase, Ypt11 is required for transport of mitochondria and the Golgi, respectively. For transport of mitochondria, both Ypt11 and a mitochondria-specific adaptor protein Mmr1 are required bind to distinct regions on Myo2 (Chernyakov et al., 2013; Eves et al., 2012; Fortsch et al., 2011; Itoh et al., 2004; Itoh et al., 2002; Lewandowska et al., 2013). Similarly, Ret1 and Ypt11 are each required for Myo2-dependent transport of the Golgi (Arai et al., 2008). Note that the binding site on Myo2 for Ret1, a coatomer subunit, is not known.

For other Myo2 cargoes, a Rab GTPase is not required. Inp2 (Fagarasanu et al., 2006) and Pex19 (Otzen et al., 2012), directly contact Myo2 on distinct sites, and are required for peroxisome transport. Vac17, for vacuole transport (Ishikawa et al., 2003; Tang et al., 2003), and Kar9, for transport of astral microtubules (Beach et al., 2000; Yin et al., 2000), each bind to distinct sites on the Myo2 cargo binding domain (Eves et al., 2012). For both of these cargoes, there is no known requirement for a Rab GTPase. Since Rab GTPases play regulatory roles via their cycling between an active GTP-bound state and inactive GDP state, the mechanisms that govern the attachment and detachment of Rab-GTPase independent cargoes, are likely distinct from cargoes where a Rab GTPase plays a role. In addition, the sites for Kar9 and Inp2 overlap with each other and with the Rab GTPase binding site, which suggests that there may be competition/coordination between secretory vesicles, mitochondria, the Golgi, peroxisomes, and astral microtubules for access to Myo2. Together, these findings indicate that the globular tail domain of Myo2 plays key roles in the regulation of its attachment to cargoes.

Cargo adaptor proteins of mammalian myosin V motors

The Rab GTPase binding site on Myo2 is conserved in myosin Va and Vb, and serves as a binding site for Rab11 (Lindsay et al., 2013; Pylypenko et al., 2016; Roland et al., 2011)}. This binding is critical for recycling from endosomes to the plasma membrane. Notably this site is not conserved in myosin Vc (Pylypenko et al., 2013). However, a nearby overlapping region on myosin Vc, which is also conserved in myosin Va, binds to Rab3A and Rab39 (Dolce et al., 2020; Lindsay et al., 2013). This site is present in myosin Va and Vc but is not conserved in myosin Vb. These distinct yet overlapping Rab GTPases binding sites in the globular tail domain likely play a role in determining which cargoes bind to different myosin V motors. In the case of myosin Va, which contains both the Rab11, Rab3A and Rab39 sites within its globular tail, these overlapping sites likely specify the attachment of selected cargoes at different times or places within a single cell.

Similar to Myo2, Rab GTPases that bind directly to the globular tail domain of mammalian myosin V motors act in concert with additional cargo adaptors. For example, Rab11 functions with the Rab11 family interacting protein 2 (FIP2). Rab11 and FIP2 both bind to the globular tail domain of myosin Vb to form a tripartite complex (Ji et al., 2019; Schafer et al., 2014; Wang et al., 2008), which is required for recycling from endosomes to the plasma membrane. An equivalent complex is also essential for rhodopsin transport and rhabdomere growth in Drosophila (Li et al., 2007), which suggests that this complex is widely conserved.

In addition to Rab GTPase sites within the globular tail domain, there are additional Rab GTPase binding sites in the upstream coiled-coil domains (Lindsay et al., 2013). Some of these Rab GTPase sites reside within tissue-specific, and cargo-specific alternatively spliced exons in myosin Va and Vb (reviewed in (Welz and Kerkhoff, 2019)). In addition, while myosin Vc contains less splice sites in this region, myosin Vc also binds selected Rab GTPases in equivalent regions of its coiled-coil domain (Bultema et al., 2014; Welz and Kerkhoff, 2019)). The sites within the coiled-coil regions, combined with sites in the globular tail, add complexity to how mammalian myosin V motors attach to cargoes.

RPGRIP1L, whose binding to myosin Va is required for ciliogenesis, also interacts with the globular tail domain of myosin Va, at a site that is conserved with the Myo2 binding sites for Inp2 and Kar9 (Assis et al., 2017). Importantly, binding to this site overlaps with, and occludes the Rab11 binding site. It is possible that attachment of RPGRIP1L to myosin Va does not require a Rab GTPase. Taken together, the diversity of cargo adaptors, as well as the multiple overlapping sites within the globular tail domain, as well as sites in the upstream coiled-coil region, play key roles in the regulation of the attachment of cargoes to myosin V motors.

Rab GTPases with roles in myosin V cargo transport that do not directly contact myosin V

Rab GTPases that do not directly contact myosin V also play essential roles in the attachment of myosin V motors to membranes. The most extensively studied is Rab27a, which has multiple functions in membrane trafficking including association with myosin V and other myosin motors, and in myosin-independent pathways (Fukuda, 2013). For myosin Va-dependent transport of melanosomes, melanophilin (also referred to as MLPH or Slac2-a) directly contacts the myosin Va globular tail, and bridges myosin Va to Rab27a, which does not directly contact myosin Va (Fukuda and Kuroda, 2002; Hume et al., 2002; Nagashima et al., 2002; Provance et al., 2002; Wu et al., 2002).

Melanophilin binds two sites on myosin Va, a site on the globular tail, and a site in melanosome-specific exon F (Yao et al., 2015). The melanophilin site on the globular tail is in a conserved region that overlaps with the Vac17 and Mmr1 sites on Myo2 (Wei et al., 2013). Similarly, Granuphilin a/b and Rabphilin3a, which function in dense core granule secretion, have structural similarity with melanophilin, and bind in the same region as melanophilin on the globular tail of myosin Va (Brozzi et al., 2012; Wei et al., 2013). In addition, Granuphilin a and Rabphilin3a binding to a brain-specific myosin Va splice isoform, require as second site, which is in exon E of the coiled-coil region. Similar with melanophilin, Granuphilin a/b and Rabphilin3a bind directly to Rab27a and Myosin Va to form tripartite complexes.

Regulation of myosin V interaction with cargo-specific adaptor proteins

Posttranslational modification of either the motor and/or cargo-specific adaptor proteins may play key roles in the initiation of cargo attachment to the motor. Indeed, cell-cycle dependent, direct phosphorylation of Vac17 by the main cyclin-dependent kinase in yeast, Cdc28/Cdk1, coordinates the initiation of vacuole transport with the cell-cycle (Peng and Weisman, 2008). Interestingly, this may be part of a feed-forward mechanism. The proper transport of the vacuole to the bud is required for the activation of Cdk1 in early G1 phase (Jin and Weisman, 2015).

Phosphorylation sites in the Myo2 globular tail domain may also be important for the association of Myo2 with Vac17 and/or other cargo adaptor proteins. Three phosphorylation sites in the globular tail of Myo2 were identified (Legesse-Miller et al., 2006), but mutation of these sites did not perturb yeast viability. However, expression of this Myo2 mutant in combination with Vac17 mutated in the four Cdk1 sites, results in a complete defect in vacuole inheritance (Peng and Weisman, 2008). One caveat of these studies is that while pull-down experiments revealed that mutation of the Cdk1 sites in Vac17 disrupted the ability of Myo2 to bind Vac17, further mutation of the Myo2 phosphorylation sites, either on their own or in combination with the Vac17 mutant missing the Cdk1 sites, surprisingly did not further impact the association of Myo2 with Vac17 (Peng and Weisman, 2008). Note that at the time of these studies, phospho-proteomics was a new area of research, thus some of the key phosphorylation sites may not have been identified.

Other cargo-specific adaptor proteins for Myo2, including Kar9, Mmr1, and Inp2 also contain predicted Cdk1 sites (Peng and Weisman, 2008), as well as phosphorylation sites for additional kinases. Importantly, Kar9 (Maekawa et al., 2003; Yin et al., 2000), Mmr1 (Swayne et al., 2011), and Inp2 (Oeljeklaus et al., 2016), are phosphorylated.

Further evidence that phosphorylation of Myo2 and/or cargo adaptor proteins regulates their association, comes from the discovery that deletion of the Ptc1 phosphatase, results in defects in Myo2-based movement of mitochondria, vacuoles, secretory vesicles, and peroxisomes (Du et al., 2006; Jin et al., 2009; Roeder et al., 1998). Furthermore, loss of Ptc1, resulted in the mis-localization of Myo2 and lowered levels of the adaptor proteins, Vac17, Kar9, and Inp2. Moreover, in vivo studies suggest that Ptc1 plays a role in the dephosphorylation of Mmr1 (Swayne et al., 2011). Together, these diverse findings suggest that further identification of phosphorylation sites on cargo-specific adaptor proteins, and the Myo2 cargo-binding domain, as well as their potential roles in the regulation of the initiation of cargo transport should be investigated.

Direct phosphorylation of the globular tail domain of mammalian myosin V regulate its association with cargoes. Phosphorylation of this region in myosin Va by Akt2, promotes GLUT4 vesicle translocation to the plasma membrane (Yoshizaki et al., 2007). Note that in studies of Xenopus, phosphorylation of this conserved site on myosin V had the opposite effect and results in the dissociation of cargoes (Karcher et al., 2001). This suggests that additional mechanisms specify whether this site has a positive or negative regulatory role in myosin V association with cargoes. Conversely, it is currently not known whether interactions of cargo-specific adaptors with mammalian myosin V are also regulated by direct phosphorylation of the adaptors.

Allosteric changes play roles in the regulation of cargo adaptor binding to mammalian myosin V motors. In vitro, the association of melanophilin with Rab27a facilitates melanophilin association with myosin Va (Fukuda and Itoh, 2004), which suggests that Rab27a may change the conformation of melanophilin to facilitate its binding to myosin Va. There is likely an ordered assembly to this tripartite complex. Rab27a is constitutively on membranes, whereas both melanophilin and myosin Va rapidly cycle on and off of melanosomes (Robinson et al., 2019). In addition, the Rab-interacting lysosomal protein-like 2 (RILPL2), interacts directly with the globular tail of myosin Va, which induces the exposure of the melanophilin binding site (Cao et al., 2019).

Similarly, allosteric changes likely impact myosin Vb association with cargoes. in hippocampal neurons, Ca++ promotes the formation of the Myosin Vb, Rab11, and FIP2 tripartite complex, and is required for LTP-induced delivery of AMPA receptors from recycling endosomes to the cell surface, as well as new dendritic spine formation (Wang et al., 2008).

Roles for cargo adaptors in the activation of myosin V

In vitro studies revealed that myosin Va motors constitutively reside in an inactive state. A major contribution to their inactivation is that the motors are folded such that the motor domain is bound to the globular tail domain (Liu et al., 2006; Thirumurugan et al., 2006). This docking may prevent the movement of the converter/lever arm during the ATP hydrolysis cycle of the motor (Li et al., 2008). Importantly, binding of the myosin Va cargo adaptor protein, melanophilin plays a role in the release of this inhibition (Cao et al., 2019; Li et al., 2005; Sckolnick et al., 2013; Yao et al., 2015). The melanophilin binding site on the globular tail is nearby, but distinct from the site on the globular tail that directly contacts the motor domain (Yao et al., 2015). Rab36 stimulates RILPL2 to interact with the myosin-Va globular tail domain, which promotes an allosteric change and exposes the melanophilin binding site. This results in the release of the motor headgroup and promotes motor activity (Cao et al., 2019).

While there are not yet in vitro studies of whether Myo2 undergoes a similar autoinhibition-activation cycle, in vivo studies suggest that Myo2 also transitions between a closed, autoinhibited state, and an open active conformation. The residues on the motor and globular tail of myosin Va that contact each other to achieve autoinhibition are conserved between mammalian myosin V motors and yeast Myo2. Mutation of the predicted contact sites on either the motor or cargo binding domain result in defects in cell growth, abnormal transport of Myo2 cargoes, and an accumulation of Myo2 at its terminal delivery sites (Donovan and Bretscher, 2015). Importantly, these defects are partially corrected in a charge reversal mutant. Defects due to mutation of the three negatively charged residues in the motor contact site to positively charged residues are partially compensated when the positively charged residues in the cargo binding domain are simultaneously changed to negatively charged residues. That Myo2 cycles through an autoinhibited state raises the question of what cargo adaptor releases the inhibition. Importantly, the in vitro studies with mammalian myosin Va, combined with in vivo studies of yeast Myo2, provide strong support for the hypothesis that autoinhibition of myosin V motors is a critical part of their regulation, and is required for normal cell function.

Cargo adaptor regulation of the actin cytoskeleton

Actin dynamics play a critical role in the regulation of myosin V movement. Evidence for the importance of dynamic rather than static filaments, came from studies that showed that stabilization of actin filaments inhibited myosin V-based melanophore movement to the cell periphery (Semenova et al., 2008). Moreover in vitro studies also revealed that the three-dimensional architecture of actin filaments plays a role in the regulation of myosin Va movement (Lombardo et al., 2019). These findings suggest that the actin filaments utilized by myosin V need to undergo dynamic remodeling during myosin V-based transport.

Notably, proteins that bind to the myosin V globular tail domain play key roles in the regulation of the actin cytoskeleton. These proteins include melanophilin, and the recently discovered actin nucleator proteins, Spire1 and Spire2 (Pylypenko et al., 2016). Melanophilin binds to filamentous actin (Fukuda and Kuroda, 2002; Kuroda et al., 2003), and addition of melanophilin in vitro increases in the number of active processive myosin Va motors (Sckolnick et al., 2013). Importantly, the addition of melanophilin also slows the rate of myosin Va transport, while increasing its run length (Sckolnick et al., 2013). These changes suggest that in addition to the release of autoinhibition, melanophilin causes a change in actin filament structure and increases the length or other properties of the actin filaments. Moreover, phosphorylation of the actin-binding domain of melanophilin by PKA positively regulates the ability of a myosin Va-melanophilin-Rab27a tripartite complex to move on actin, and negatively regulates the association of the complex with microtubules (Oberhofer et al., 2017). These findings support the hypothesis that melanophilin association with actin is regulated, that melanophilin regulates actin dynamics and that this additional function of melanophilin plays an important role in myosin Va transport.

Recent studies revealed that Spire1/2 is critical for the proper distribution of melanosomes (Alzahofi et al., 2020). Notably, Spire1/2 binds directly to the globular tail of myosin Va in a site that overlaps with the melanophilin site (Pylypenko et al., 2016). Spire1/2 also binds to the actin nucleator, Formin2. Spire1/2 and Rab11 bind distinct sites on the myosin Va globular tail, and the three proteins form a tripartite complex. Moreover, the structure of the Spire1/2-myosin Va complex suggests that similar to melanophilin, Spire1/2 may activate myosin V by opening the autoinihibited state. Importantly, the Spire1/2 binding site is conserved in myosin Vb and Vc. Thus, Spire1/2 may function in the activation of all three mammalian myosin V motors.

In addition to myosin V interaction with the actin nucleating protein Spire, myosin Va directly binds to the actin disassembly protein, MICAL1 (Niu et al., 2020). MICAL1 controls actin disassembly via oxidation of actin (Fremont et al., 2017). The myosin V binding site for MICAL1 is conserved in myosin Va, Vb, and Vc, and notably overlaps with the melanophilin and Spire1/2 binding sites. Importantly, MICAL1 binding to myosin Va is essential for the proper completion of abscission during cytokinesis, and the release of myosin Va from Rab11 (Niu et al., 2020). One interpretation of these observations is that MICAL1 plays a direct role in the unloading of myosin V cargoes. Alternatively, or in addition, MICAL1 may halt myosin V movement via disassembly of the actin filament tracks, which may signal to a cargo detachment pathway to unload the cargo. While MICAL1 has only been shown to function with myosin Va in cell abscission, given that MICAL1 binds both myosin Va and Vb in vitro, and that the site is conserved in all mammalian myosin V motors, it is tempting to speculate that MICAL1 has a general role in late steps of myosin V-based transport. Multiple studies of mammalian myosin V reveal that actin dynamics are crucial for motor transport, and that these dynamics are achieved by at least three proteins that bind directly to the globular tail domain. However, it is not known whether yeast Myo2 participates in similar pathways.

Roles of cargo adaptors in the regulated detachment of myosin V motors from cargoes.

The initial focus on molecular motor-based transport of cargoes, was devoted to understanding how cargoes were selected and loaded onto the motor. However, several studies have revealed that once cargoes arrive at their destination, there are ordered pathways to dissociate the cargoes from their motors. These pathways may target the motor, the cargo adaptor, and/or the cytoskeleton (see (Niu et al., 2020)).

Studies of Xenopus melanophores revealed that the cell-cycle dependent phosphorylation of the globular tail domain of myosin V is required for its dissociation from melanosomes (Karcher et al., 2001; Rogers et al., 1999). These findings indicate that specific pathways are required for the release of myosin V from cargoes, and that modulation of the cargo binding domain of myosin V plays a role in the dissociation of at least one type of cargo.

Myo2 dissociation from the vacuole requires the convergence of two parallel pathways that each act directly on the vacuole specific adaptor, Vac17 (Wong et al., 2020). One pathway involves the ubiquitylation of Vac17 in its PEST sequence (Tang et al., 2003; Yau et al., 2014). Early in transport, an inactive E3 ubiquitin ligase binds to the Vac17-PEST sequence (Yau et al., 2014), and then becomes activated at the destination – the bud cortex (Yau et al., 2017). A kinase at the bud cortex phosphorylates Vac17 in its PEST sequence, which results in the activation of the E3 ligase and ubiquitylation of Vac17 (Yau et al., 2017). A parallel pathway initiates on the vacuole, and results in the phosphorylation of Vac17 in the Myo2 binding domain, and in an as yet unidentified mechanism that includes the conserved proteins Vps41 and Yck3, results in the extraction of Vac17 from the Myo2 complex, and degradation of Vac17 by the proteasome (Wong et al., 2020). That parallel pathways are required, underscores the importance of regulating the detachment of cargoes from molecular motors. Premature detachment would result in a defect in the arrival of the cargo at its destination, whereas delayed detachment could potentially drag the cargo to other itineraries of the motor, as is observed for the yeast vacuole (Tang et al., 2003; Wong et al., 2020; Yau et al., 2014; Yau et al., 2017).

There are indications that release of some other cargoes from myosin V motors are also regulated by pathways that are similar to those that act on the dissociation of Vac17 from Myo2. For example, dissociation of Kar9 from Myo2 requires ubiquitylation of Kar9 followed by its degradation by the proteasome. Note that Kar9 ubiquitylation requires phosphorylation by Cdk1 (Kammerer et al., 2010). In addition, melanophilin has PEST sequences, and mutation of one of its PEST sequence results in a defect in melanosome cellular distribution consistent with a defect in melanophilin detachment from myosin Va (Fukuda and Itoh, 2004). In addition, Vps41 and Yck3 (a Casein Kinase I) are conserved in yeast and mammals, and found in all cell types. This raises the possibility that the Vps41- and Yck3- dependent pathway may be conserved for myosin V dissociation from additional cargoes.

Degradation of cargo adaptors may not be involved in the disengagement of myosin V from some cargoes. In the case of Myo2 delivery of secretory vesicles, the release of Myo2 occurs at the bud cortex, and requires GTP hydrolysis of Sec4 and exocyst function, but not t-SNARE function (Donovan and Bretscher, 2012). These findings suggest that release of Myo2 is coupled to a step immediately following secretory vesicle tethering. While it remains an open possibility that other proteins are also required, the fact that the Rab GTPase Sec4 directly contacts the Myo2 globular tail domain, and that Sec4 undergoes major conformational changes during GTP hydrolysis (Stroupe and Brunger, 2000), suggests that GTP hydrolysis of Sec4 could be the main factor in the release of Myo2 from secretory vesicles.

Conclusion

Future studies will likely reveal more complete mechanisms for the activation and inactivation of myosin V and the relationship of this cycle to the regulation of the attachment and detachment of cargoes. This is a relatively unexplored area of research. Thus, it is likely that additional common principles for mechanisms that govern the regulation of myosin V will be discovered. For example, similar to mammalian myosin V, do cargo adaptors of yeast Myo2 directly regulate actin filaments? For all myosin V motors, are the delivery of each type of cargo coordinated with all cargoes carried by that type of motor? What are the signaling pathways that integrate motor activity with motor-cargo interactions, and how are these pathways integrated with the cellular pathways that require myosin V-dependent transport. Future studies will likely reveal answers to these questions.