The Src Homology Domain 3 (SH3) of a Yeast Type I Myosin, Myo5p, Binds to Verprolin and Is Required for Targeting to Sites of Actin Polarization

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Δ myo5Δ cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A–induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37°C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Δ myo5Δ cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Δ myo5Δ cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.     

Complex formation with Ypt11p,a rab-type small GTPase,is essential to facilitate the function of Myo2p,a class V myosin,in mitochondrial distribution in Saccharomyces cerevisiae

We identified Ypt11p, a rab-type small GTPase, by its functional and two-hybrid interaction with Myo2p, a class V myosin of the budding yeast Saccharomyces cerevisiae. The tail domain of Myo2p was coimmunoprecipitated with Ypt11p, suggesting that Ypt11p forms a complex with Myo2p at its tail domain in vivo. Mutational analysis of YPT11 suggests that Myo2p is a putative effector of Ypt11p. Deletion of YPT11 induced partial delay of mitochondrial transmission to the bud, and overexpression of YPT11 resulted in mitochondrial accumulation in the bud, indicating that Ypt11p acts positively on mitochondrial distribution toward the bud. We isolated two myo2 mutants, myo2-338 and myo2-573, which showed genetic interactions with YPT11. The myo2-573 mutation, identified by a synthetic lethal interaction with ypt11-null, induced a defect in mitochondrial distribution toward the bud, indicating that Myo2p plays a crucial role in polarized distribution of mitochondria. The myo2-338 mutation was identified as the mutation that abolished the effect of overexpressed YPT11, such as the Ypt11p-dependent accumulation of mitochondria in the bud, and the affinity of Myo2p for Ypt11p was reduced. These results indicate that complex formation of Ypt11p with Myo2p accelerates the function of Myo2p for mitochondrial distribution toward the bud.     

The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.     

The yeast kinesin-related protein Smy1p exerts its effects on the class V myosin Myo2p via a physical interaction

We have discovered evidence for a physical interaction between a class V myosin, Myo2p, and a kinesin-related protein, Smy1p, in budding yeast. These proteins had previously been linked by genetic and colocalization studies, but we had been unable to determine the nature of their association. We now show by two-hybrid analysis that a 69-amino acid region of the Smy1p tail interacts with the globular portion of the Myo2p tail. Deletion of this myosin-binding region of Smy1p eliminates its ability to colocalize with Myo2p and to overcome the myo2-66 mutant defects, suggesting that the interaction is necessary for these functions. Further insights about the Smy1p-Myo2p interaction have come from studies of a new mutant allele, myo2-2, which causes a loss of Myo2p localization. We report that Smy1p localization is also lost in the myo2-2 mutant, demonstrating that Smy1p localization is dependent on Myo2p. We also found that overexpression of Smy1p partially restores myo2-2p localization in a myosin-binding region-dependent manner. Thus, overexpression of Smy1p can overcome defects in both the head and tail domains of Myo2p (caused by the myo2-66 and myo2-2 alleles, respectively). We propose that Smy1p enhances some aspect of Myo2p function, perhaps delivery or docking of vesicles at the bud tip.     

Two distinct regions in a yeast myosin-V tail domain are required for the movement of different cargoes

The Saccharomyces cerevisiae myosin-V, Myo2p, is essential for polarized growth, most likely through transport of secretory vesicles to the developing bud. Myo2p is also required for vacuole movement, a process not essential for growth. The globular region of the myosin-V COOH-terminal tail domain is proposed to bind cargo. Through random mutagenesis of this globular tail, we isolated six new single point mutants defective in vacuole inheritance, but not polarized growth. These point mutations cluster to four amino acids in an 11-amino acid span, suggesting that this region is important for vacuole movement. In addition, through characterization of myo2-DeltaAflII, a deletion of amino acids 1,459-1,491, we identified a second region of the globular tail specifically required for polarized growth. Whereas this mutant does not support growth, it complements the vacuole inheritance defect in myo2-2 (G1248D) cells. Moreover, overexpression of the myo2-DeltaAflII globular tail interferes with vacuole movement, but not polarized growth. These data indicate that this second region is dispensable for vacuole movement. The identification of these distinct subdomains in the cargo-binding domain suggests how myosin-Vs can move multiple cargoes. Moreover, these studies suggest that the vacuole receptor for Myo2p differs from the receptor for the essential cargo.     

Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.     

Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp

Fission yeast myo1(+) encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1(+) is not lethal, Deltamyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1(+) is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Deltamyo1 defects. However, it fails to rescue the Deltamyo1 Deltawsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.     

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S(1518)A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S(1518)E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25 degrees C but not at 36 degrees C. GFP-Myo2S(1518)E rings persisted at 36 degrees C in cdc7-24 but not in another SIN kinase mutant, sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy of myo2(+) was fused to GFP (strain myo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36 degrees C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.     

Mmr1p is a mitochondrial factor for Myo2p-dependent inheritance of mitochondria in the budding yeast

Class V myosins play a pivotal role in organelle distribution. In the budding yeast, Myo2p, a class V myosin, is essential for mitochondrial distribution. We identified MMR1 as a high-dose suppressor of the myo2 mitochondrial defect and that Mmr1p resides restrictively on the bud-localizing mitochondria and forms a complex with Myo2p tail. Mmr1p loss delayed mitochondrial transfer to buds and completely abolished mitochondrial distribution in the absence of Ypt11p, which promotes mitochondrial distribution by complex formation with Myo2p tail. The myo2-573 mutation, which causes a mitochondrial distribution defect and inactivates the Mmr1p function, reduced association between Myo2p and Mmr1p and depolarized Mmr1p localization on mitochondria. These strongly suggest that Mmr1p is a key mitochondrial component of the link between Myo2p and mitochondria for Myo2p-dependent mitochondrial distribution. Genetical analysis revealed that the Mmr1p-Myo2p pathway is independent of the Ypt11p-Myo2p pathway, suggesting that an essential system for mitochondrial distribution is composed of two independent Myo2p pathways.     

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.     

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.     

A critical role for the type V myosin, Myo52, in septum deposition and cell fission during cytokinesis in Schizosaccharomyces pombe

Cytokinesis in fission yeast involves the coordination of septum deposition with the contraction of a cytokinetic actomyosin ring. We have examined the role of the type V myosin Myo52 in the coupling of these two events by the construction of a series of deletion mutants of the Myo52 tail and a further mutant within the ATP binding domain of the head. Each mutant protein was ectopically expressed in fission yeast cells. Each truncation was assayed for the ability to localize to the cell poles and septum (the normal cellular locations of Myo52) and to rescue the morphology defects and temperature sensitivity of a myo52Delta strain. A region within the Myo52 tail (amino acids 1320-1503), with a high degree of similarity to the vesicle-binding domain of the budding yeast type V myosin Myo2p, was essential for Myo52's role in the maintenance of growth polarity and cell division. A separate region (amino acids 1180-1320) was required for Myo52 foci to move throughout the cytoplasm; however, constructs lacking this region, but which retained the ability to dimerize still associated with actin at sites of cell growth. Not all of the Myo52 truncations which localized rescued the morphological defects of myo52Delta, demonstrating that loss of function was not simply brought about by an inability of mutant proteins to target the correct cellular location. By contrast, Myo52 motor activity was required for both localization and cellular function. myo52Delta cells were unable to efficiently localize the beta-1,3-glucan synthase, Bgs1, either at the cell poles or at the division septum, regions of cell wall deposition. Bgs1 and Myo52 localized to vesicle-like dots at the poles in interphase and these moved together to the septum at division. These data have led to the formulation of a model in which Myo52 is responsible for the delivery of Bgs1 and associated molecules to polar cell growth regions during interphase. On the commencement of septum formation, Myo52 transports Bgs1 to the cell equator, thus ensuring the accurate deposition of beta-1,3-glucan at the leading edge of the primary septum.     

Cytokinesis depends on the motor domains of myosin-II in fission yeast but not in budding yeast

Budding yeast possesses one myosin-II, Myo1p, whereas fission yeast has two, Myo2p and Myp2p, all of which contribute to cytokinesis. We find that chimeras consisting of Myo2p or Myp2p motor domains fused to the tail of Myo1p are fully functional in supporting budding yeast cytokinesis. Remarkably, the tail alone of budding yeast Myo1p localizes to the contractile ring, supporting both its constriction and cytokinesis. In contrast, fission yeast Myo2p and Myp2p require both the catalytic head domain as well as tail domains for function, with the tails providing distinct functions (Bezanilla and Pollard, 2000). Myo1p is the first example of a myosin whose cellular function does not require a catalytic motor domain revealing a novel mechanism of action for budding yeast myosin-II independent of actin binding and ATPase activity.     

Cell polarity protein Spa2P associates with proteins involved in actin function in Saccharomyces cerevisiae

Spa2p is a nonessential protein that regulates yeast cell polarity. It localizes early to the presumptive bud site and remains at sites of growth throughout the cell cycle. To understand how Spa2p localization is regulated and to gain insight into its molecular function in cell polarity, we used a coimmunoprecipitation strategy followed by tandem mass spectrometry analysis to identify proteins that associate with Spa2p in vivo. We identified Myo1p, Myo2p, Pan1p, and the protein encoded by YFR016c as proteins that interact with Spa2p. Strikingly, all of these proteins are involved in cell polarity and/or actin function. Here we focus on the functional significance of the interactions of Spa2p with Myo2p and Myo1p. We find that localization of Spa2GFP to sites of polarized growth depends on functional Myo2p but not on Myo1p. We also find that Spa2p, like Myo2p, cosediments with F-actin in an ATP-sensitive manner. We hypothesize that Spa2p associates with actin via a direct or indirect interaction with Myo2p and that Spa2p may be involved in mediating polarized localization of polarity proteins via Myo2p. In addition, we observe an enhanced cell-separation defect in a myo1spa2 strain at 37 degrees C. This provides further evidence that Spa2p is involved in cytokinesis and cell wall morphogenesis.     

Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo

The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1—which binds Myo2—its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo.     

The class V myosin motor protein, Myo2, plays a major role in mitochondrial motility in Saccharomyces cerevisiae

The actin cytoskeleton is essential for polarized, bud-directed movement of cellular membranes in Saccharomyces cerevisiae and thus ensures accurate inheritance of organelles during cell division. Also, mitochondrial distribution and inheritance depend on the actin cytoskeleton, though the precise molecular mechanisms are unknown. Here, we establish the class V myosin motor protein, Myo2, as an important mediator of mitochondrial motility in budding yeast. We found that mutants with abnormal expression levels of Myo2 or its associated light chain, Mlc1, exhibit aberrant mitochondrial morphology and loss of mitochondrial DNA. Specific mutations in the globular tail of Myo2 lead to aggregation of mitochondria in the mother cell. Isolated mitochondria lacking functional Myo2 are severely impaired in their capacity to bind to actin filaments in vitro. Time-resolved fluorescence microscopy revealed a block of bud-directed anterograde mitochondrial movement in cargo binding-defective myo2 mutant cells. We conclude that Myo2 plays an important and direct role for mitochondrial motility and inheritance in budding yeast.     

Yeast Rgd3 is a phospho-regulated F-BAR–containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

Polarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Overexpression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR (Fes/CIP4 homology-Bin-Amphiphysin-Rvs protein)-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 overexpression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP₂-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho state, and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.     

Role of fission yeast myosin I in organization of sterol-rich membrane domains

Specialized membrane domains containing lipid rafts are thought to be important for membrane processes such as signaling and trafficking. An unconventional type I myosin has been shown to reside in lipid rafts and function to target a disaccharidase to rafts in brush borders of intestinal mammalian cells. In the fission yeast Schizosaccharomyces pombe, distinct sterol-rich membrane domains are formed at the cell division site and sites of polarized cell growth at cell tips. Here, we show that the sole S. pombe myosin I, myo1p, is required for proper organization of these membrane domains. myo1 mutants lacking the TH1 domain exhibit a uniform distribution of sterol-rich membranes all over the plasma membrane throughout the cell cycle. These effects are independent of endocytosis because myo1 mutants exhibit no endocytic defects. Conversely, overexpression of myo1p induces ectopic sterol-rich membrane domains. Myo1p localizes to nonmotile foci that cluster in sterol-rich plasma membrane domains and fractionates with detergent-resistant membranes. Because the myo1p TH1 domain may bind directly to acidic phospholipids, these findings suggest a model for how type I myosin contributes to the organization of specialized membrane domains.     

Cdc50p,a conserved endosomal membrane protein,controls polarized growth in Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and the growth of cell surface are polarized, mediating bud emergence, bud growth, and cytokinesis. We identified CDC50 as a multicopy suppressor of the myo3 myo5-360 temperature-sensitive mutant, which is defective in organization of cortical actin patches. The cdc50 null mutant showed cold-sensitive cell cycle arrest with a small bud as reported previously. Cortical actin patches and Myo5p, which are normally localized to polarization sites, were depolarized in the cdc50 mutant. Furthermore, actin cables disappeared, and Bni1p and Gic1p, effectors of the Cdc42p small GTPase, were mislocalized in the cdc50 mutant. As predicted by its amino acid sequence, Cdc50p appears to be a transmembrane protein because it was solubilized from the membranes by detergent treatment. Cdc50p colocalized with Vps21p in endosomal compartments and was also localized to the class E compartment in the vps27 mutant. The cdc50 mutant showed defects in a late stage of endocytosis but not in the internalization step. It showed, however, only modest defects in vacuolar protein sorting. Our results indicate that Cdc50p is a novel endosomal protein that regulates polarized cell growth.     

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.