Mammals have three myosin V motors: myosin Va, myosin Vb, and myosin Vc (
28). Myosin Va is the best characterized mammalian myosin V. In mouse, myosin Va is encoded by the dilute locus. The dilute mutants have a lightened coat color, and null mutants die of a neurological disorder within a few weeks of birth (
32). Similarly, mutations in the human ortholog of dilute cause Griscelli's syndrome, a rare recessive disease characterized by neurological and pigmentation defects (
31,
36). Several studies have demonstrated that myosin Va is important for melanosome transport in melanocytes (
34,
37,
39,
49,
51) and transport of smooth endoplasmic reticulum in Purkinje cells (
46). In addition, myosin Va moves chromaffin vesicles (
41) and membranous vesicles in nerve cells (
5,
12). While these myosin Va studies report on the movement of a single cargo within a selected cell type, it is likely that myosin Va moves multiple cargoes within a single type of cell (
9).
Myosin V moves via attachment of its amino terminal head (motor) domain to actin cables; its carboxyl terminal tail domain anchors it to cargoes via attachments to organelle-specific receptors. Individual myosin V motors move multiple cargoes, where the cargoes move to distinct locations at different times. Both the spatial and temporal regulation of movement of an individual cargo occurs in part via organelle-specific receptors. To date, a few organelle-specific myosin V receptors have been identified. The melanosome-specific myosin Va receptor is composed of melanophilin, which binds directly to myosin Va and simultaneously to Rab27a (
52). Similarly, the myosin Vb globular tail interacts with Rab11a and the Rab11 family interacting protein 2 (
15). The vacuole-specific myosin V receptor is composed of Vac17p, which binds directly to yeast Myo2p and simultaneously to the vacuole membrane protein Vac8p (
6,
20,
47). In a separate functional complex, the Myo2p globular tail binds directly to Kar9p, which in turn binds to the microtubule end binding protein, Bim1p/EB1 (
1,
53).
Notably, distinct regions of the myosin V globular tail appear to bind different organelle-specific receptors. For example, seven single point mutations in the Myo2p globular tail were isolated that cause a vacuole or lysosome inheritance defect without affecting secretory vesicle movement (
7,
8). Six of these mutations are single amino acid changes at D1297, L1301, N1304, or N1307. These amino acids lie along a face of a predicted α-helix (
7). Subsequent studies have strongly suggested that these residues directly bind to the vacuole-specific receptor, Vac17p (
6,
20). Likewise, point mutations that were identified in the globular tail of mouse myosin Va (I1510N, M1513K, or D1519G) appear to specifically affect melanosome movement (
18). These mutations were later found to partially impair the binding of the myosin Va globular tail to Slac2-a/melanophilin (
13).
Conversely, mutations in the Myo2p globular tail have been identified that affect secretory vesicle movement without affecting vacuole movement. Several conditional lethal
myo2 alleles, which result from point mutations in the globular tail, do not affect the ability of Myo2p to move the vacuole. However, at the nonpermissive temperature, these mutant alleles are defective in secretory vesicle movement (
44). Likewise,
myo2-Δ
AflII, which contains a small deletion in the Myo2p globular tail, cannot support yeast viability, presumably due to a defect in moving secretory vesicles; however,
myo2-Δ
AflII functions in vacuole movement (
7). Analysis of the above vacuole-specific and secretory vesicle-specific point mutants demonstrates that the Myo2p globular tail can be divided into a vacuole-specific region and secretory vesicle-specific region (
7,
44).
These studies led to the hypothesis that the myosin V globular tail itself plays a regulatory role in specifying cargoes. Specifically, we speculate that occupancy of subdomain II by a cargo might block the binding of a cargo to subdomain I. Here we focus on a point mutant which may represent the proposed inactive conformation of subdomain I. myo2-2(G1248D) lies outside of the region that binds directly to Vac17p. Moreover, unlike the vacuole-specific point mutations at residues that bind directly to Vac17p (D1297, L1301, N1304, or N1307), myo2-2 has multiple defects. Multiple intragenic suppressors of myo2-2 were isolated and characterized. Analysis of myo2-2 and its suppressors strongly suggests that G1248 and the surrounding region may contribute to forming both a “closed or inactive” and “open or active” conformation of subdomain I.
DISCUSSION
Myosin V molecular motors transport multiple cargoes to distinct places at different times. Therefore, there is likely to be stringent regulation of when myosin V moves an individual cargo. This regulation could occur either via the activation or deactivation of a motor protein that is constitutively attached to its cargo and/or via the attachment or detachment of the motor protein from a specific cargo. Multiple lines of evidence indicate that this latter type of regulation occurs. For example, melanosome transport in
Xenopus laevis melanophores is regulated by reversible association with myosin V in a cell cycle-coordinated manner (
40). In addition, the discovery of cargo-specific receptor complexes (
6,
20,
52,
53) and the fact that regulated degradation of the myosin V receptor directs vacuole movement in yeast (
47) clearly indicate that this type of regulatory mechanism occurs. Note that these findings do not exclude an additional regulatory mechanism involving the activation of the motor domain.
Cargoes bind to the globular tail domain of myosin V, and it is likely that conformational changes within this domain play an active role in regulating cargo attachment (
33). Furthermore, phosphorylation of the myosin V globular tail results in its release from melanosomes (
26); this phosphorylation may act by inducing conformational changes. In addition to a role in specifying cargo, the globular tail of myosin V may also play a role in regulating myosin motor activity. It has recently been shown that myosin Va exists in two conformations, which are regulated by calcium and calmodulin. In the folded, inactive conformation of full-length myosin V, the globular tail interacts with the motor domain, while in the open, extended conformation the motor domain is free of the globular tail and is active (
27,
48). Conformational changes within the globular tail itself may be part of the regulation of motor activity.
Our recent finding that the myosin V globular tail consists of two tightly associated subdomains (
35) suggests at least two possible types of conformational changes that could play a role in specifying cargo. First, the regulation of cargo attachment could occur via a reversible interaction between the two subdomains, where some cargoes bind to the globular tail in an extended conformation, while others bind to the tail in a closed conformation. In an alternative model, the two subdomains may always be tightly associated. In this latter model, binding of cargo to one subdomain may induce a conformational change in the other subdomain that would preclude the binding of additional types of cargoes. This latter model, where subdomains I and II are always tightly associated with each other, is more likely. Note that in virtually all partially functional alleles of Myo2p, subdomain I and II interactions were similar to those observed in wild type. Moreover, in order to bind to the globular tail, the binding proteins tested required the presence of both subdomains I and II.
If the occupation of a receptor-binding site on one subdomain precludes the binding of receptors to the other subdomain, then it may be possible to identify point mutations that cause a subdomain to be constitutively locked in the conformation that prevents the binding of a subset of cargoes. Analysis of myo2-2(G1248D) strongly suggests that G1248D may be this type of point mutation.
Two lines of evidence support the hypothesis that the
myo2-2 mutation causes a conformational change that prevents the binding of a subset of cargoes. First, while secretory vesicle movement (associated with subdomain II) is normal in
myo2-2, this mutant has multiple, severe, nonrelated defects. The
myo2-2 mutant is defective in vacuole inheritance due to an inability to interact with Vac17p, the vacuole-specific Myo2p receptor. The Vac17p binding site has been mapped to subdomain I. Furthermore, the globular tail of
myo2-2 cannot interact with Smy1p. Based on the finding that deletion of the last 117 residues of the Myo2p tail abolished Myo2p-Smy1p interactions, the binding site for Smy1p had been proposed to map to the C terminus (subdomain II) (
2). However, similar truncations also abolish the ability of Myo2p to interact with Vac17p in a yeast two-hybrid test (
6,
20,
35); the primary defect in these truncations is likely due to a loss of interaction between subdomains I and II, rather than the loss of a receptor binding site (see below for further discussion). Thus, the binding region for Smy1p is unknown and may reside in either or both subdomains.
myo2-2 is also defective in nuclear spindle orientation due to an inability to bind to Kar9p; the binding region for Kar9p is also unknown.
In addition to the above defects, myo2-2p does not properly concentrate at sites of polarized growth (
7,
8). The binding partner or molecular mechanism required for Myo2p concentration is unknown.
The multiple defects observed in
myo2-2 do not appear to result from a global instability of the mutant protein. First, in cell extracts, Myo2p and myo2-2p are present at the same steady-state levels (
8). Moreover, while the
myo2-2 mutant has multiple defects, the ability of myo2-2p to move some cargoes is the same as that observed for the wild-type protein. This is most obvious for secretory vesicle movement, where a block would cause cell death and a partial block in movement would result in slower than normal growth. Notably,
myo2-2 is viable over a wide range of temperatures (18 to 37°C) (
8), and we were unable to identify any growth conditions where growth of the
myo2-2 mutant was more severely affected than growth of wild-type cells (data not shown).
In contrast to the multiple defects observed in
myo2-2, the globular tail mutations at residues D1297, L1301, N1304, or N1307 are more specifically defective in vacuole inheritance. These residues likely reside within a region of the globular tail that directly interacts with Vac17p (
6,
20). Thus, the
myo2-2 mutant appears to be unique among mutants identified to date that are defective in vacuole movement.
A second line of evidence suggesting that the globular tail of myo2-2 may be in an altered, closed or partially nonfunctional conformation is our finding that there are reproducible differences in the rate and extent of mild proteolysis of the wild-type globular tail compared with the myo2-2p globular tail.
If the
myo2-2 mutation causes a regulatory conformational change that simultaneously affects multiple cargoes, then it may be possible to identify second site mutations that simultaneously restore all Myo2p-related functions. Therefore, we screened for second site mutations that specifically restore vacuole inheritance to close to wild-type levels and then subsequently analyzed these alleles to determine whether they also suppressed other defects associated with
myo2-2. Notably, 3 of the 10 suppressors identified solely based on their ability to restore vacuole inheritance also restored interaction with Smy1p and Kar9p (Tables
3 and
4).
To date, secretory vesicles are the only known essential cargo moved by Myo2p. While Myo2p is also involved in the inheritance of mitochondria (
21,
22), an essential organelle, other mechanisms also play a role in mitochondrial movement (
4). In addition, Myo2p moves vacuoles, peroxisomes, and the late Golgi; if Myo2p-based movement of these organelles to the bud is blocked, then the organelles appear in the bud by unknown, independent processes.
Notably, the Myo2p globular tail binding region for secretory vesicles resides within subdomain II. This raises an interesting possibility that the binding sites for nonessential cargoes may reside within subdomain I, while the binding site for secretory vesicles is localized within subdomain II. If occupation of subdomain II by the putative secretory vesicle receptor inhibits cargo binding to subdomain I, this could provide a mechanism for secretory vesicle movement to take precedence over the nonessential Myo2p-based movement of other cargoes. Unfortunately, it is not yet possible to test this aspect of the model. While a region on the globular tail that is part of the secretory vesicle binding site has been mapped, the identity of the secretory vesicle-specific receptor has not yet been determined. This makes it impossible to design experiments that would mimic occupancy of the secretory vesicle binding site on subdomain II.
The binding sites for Vac17p, Smy1p, and Kar9p are not identical; mutations at residues D1297, L1301, N1304, or N1307 have little to no effect on the ability of Myo2p to interact with Smy1p or Kar9p (Tables
3 and
4). However, despite the fact that the binding sites for these proteins are not identical, the binding sites for Vac17p, Kar9p, and Smy1p could potentially overlap at Myo2p residue G1248. If the binding sites overlap, then an alternative possibility is that the global suppressors restore the binding site that these proteins share in common. However, it is not yet possible to test this hypothesis. Mapping the binding sites on Myo2p for Kar9p and Smy1p will be complex, because mutations that either interfere with subdomain I and II interactions or directly reside in the organelle-specific receptor binding site will abolish function. Indeed, either the G1248D mutation within subdomain I or the ΔAflII (residues 1459 to 1491) deletion within subdomain II blocked the ability of the Myo2p globular tail to interact with either Kar9p or Smy1p (Table
3). Identification of the precise binding sites in Myo2p for Smy1p, Kar9p, and other as yet unknown binding partners will clarify whether these sites are structurally separated yet share an overlapping regulatory region or whether the binding sites overlap.
While overlapping binding sites are possible, we favor the idea that the defect in myo2-2 may be due to a conformational defect in subdomain I that affects binding to several organelle-specific receptors. Determination and comparison of the three-dimensional structures of the globular tail from wild type versus myo2-2 may provide insights into conformational changes that contribute to the regulation and function of the globular tail.