FRET peptides reveal differential proteolytic activation in intraerythrocytic stages of the malaria parasites Plasmodium berghei and Plasmodium yoelii

Malaria is still a major health problem in developing countries. It is caused by the protist parasite Plasmodium, in which proteases are activated during the cell cycle. Ca(2+) is a ubiquitous signalling ion that appears to regulate protease activity through changes in its intracellular concentration. Proteases are crucial to Plasmodium development, but the role of Ca(2+) in their activity is not fully understood. Here we investigated the role of Ca(2+) in protease modulation among rodent Plasmodium spp. Using fluorescence resonance energy transfer (FRET) peptides, we verified protease activity elicited by Ca(2+) from the endoplasmatic reticulum (ER) after stimulation with thapsigargin (a sarco/endoplasmatic reticulum Ca(2+)-ATPase (SERCA) inhibitor) and from acidic compartments by stimulation with nigericin (a K(+)/H(+) exchanger) or monensin (a Na(+)/H(+) exchanger). Intracellular (BAPTA/AM) and extracellular (EGTA) Ca(2+) chelators were used to investigate the role played by Ca(2+) in protease activation. In Plasmodium berghei both EGTA and BAPTA blocked protease activation, whilst in Plasmodium yoelii these compounds caused protease activation. The effects of protease inhibitors on thapsigargin-induced proteolysis also differed between the species. Pepstatin A and phenylmethylsulphonyl fluoride (PMSF) increased thapsigargin-induced proteolysis in P. berghei but decreased it in P. yoelii. Conversely. E64 reduced proteolysis in P. berghei but stimulated it in P. yoelii. The data point out key differences in proteolytic responses to Ca(2+) between species of Plasmodium. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16D), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16D mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H?-ATPase (VATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1D or vma2D) or membrane domain (vph1D or vma3D) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.

Characterization of high osmolarity-induced vacuole fragmentation in "Saccharomyces c."

La majorité des organelles d'une cellule adaptent leur nombre et leur taille pendant les processus de division cellulaire, de trafic vésiculaire ou suite à des changements environnementaux par des processus de fusion et de fragmentation membranaires. Ceci est valable notamment pour le golgi, les mitochondries, les péroxisomes et les lysosomes. La vacuole est le compartiment terminal de la voie endocytaire dans la levure Saccharomyces cerevisiae\ elle correspond aux lysosomes des cellules mammifères. Suite à un choc hyperosmotique, la vacuole se fragmente en plusieurs petites vésicules. Durant ce projet, cette fragmentation a été étudiée en utilisant la technique de microscopie confocale in vivo. J'ai observé que la division de la vacuole se produit d'une façon asymétrique. La première minute après le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase est dépendante de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que d'un gradient transmembranaire de protons. Pendant les 10-15 minutes qui suivent, des vésicules se détachent dans les régions où l'on observe les invaginations pendant la phase initiale. Cette deuxième phase qui mène à la fission des nouveaux compartiments vacuolaires dépend de la production du lipide PI(3,5)P2 par la protéine Fab1. J'ai établi la suite des événements du processus de fragmentation des vacuoles et propose la possibilité d'un rôle régulateur de la protéine kinase cycline-dépendante Pho85.¦En outre, j'ai tenté d'éclaircir plus spécifiquement le rôle de Vps1 pendant la fusion et fission des vacuoles. J'ai trouvé que tous les deux processus sont dépendants de l'activité GTPase de cette protéine. De plus l'association avec la membrane vacuolaire paraît régulée par le cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'un autre facteur protéinique, ce qui permet de conclure à une interaction directe avec des lipides de la membrane. Cette interaction est au moins partiellement effectuée par le domaine GTPase, ce qui est une nouveauté pour un membre de cette famille de protéines. Une deuxième partie de Vps1, nommée insert B, est impliquée dans la liaison à la vacuole, soit par interaction directe avec la membrane, soit par régulation du domaine GTPase. En assumant que Vps1 détienne deux régions capables de liaison aux membranes, je conclus qu'elle pourrait fonctionner comme facteur de « tethering » lors de la fusion des vacuoles.¦-¦La cellule contient plusieurs sous-unités, appelées organelles, possédant chacune une fonction spécifique. Dépendant des processus qui s'y déroulent à l'intérieur, un environnement chimique spécifique est requis. Pour maintenir ces différentes conditions, les organelles sont séparées par des membranes. Lors de la division cellulaire ou en adaptation à des changements de milieu, les organelles doivent être capables de modifier leur morphologie. Cette adaptation a souvent lieu par fusion ou division des organelles. Le même principe est valable pour la vacuole dans la levure. La vacuole est une organelle qui sert principalement au stockage des aliments et à la dégradation des différents composants cellulaires. Alors que la fusion des vacuoles est un processus déjà bien décrit, la fragmentation des vacuoles a jusqu'ici été peu étudiée. Elle peut être induit par un choc osmotique: à cause de la concentration de sel élevé dans le milieu, le cytosol de la levure perd de l'eau. Par un flux d'eau de la vacuole au cytosol, la cellule est capable d'équilibrer celui-ci. Quand la vacuole perd du volume, elle doit réadapter le rapport entre surface membranaire et volume, ce qui se fait efficacement par une fragmentation d'une grande vacuole en plusieurs petites vésicules. Comment ce processus se déroule d'un point de vue morphologique n'a pas été décrit jusqu'à présent. En analysant la fragmentation vacuolaire par microscopie, j'ai trouvé que celle-ci se déroule en deux phases. Pendant la première minute suivant le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase dépend de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que du gradient transmembranaire de protons. Ce gradient s'établit par une pompe membranaire, la V-ATPase, qui transporte des protons dans la vacuole en utilisant l'énergie libérée par hydrolyse d'ATP. Après cette phase initiale, la formation de nouvelles vésicules vacuolaires dépend de la synthèse du lipide PI(3,5)P2.¦Dans la deuxième partie de l'étude, j'ai tenté de décrire comment Vps1 lie la membrane pour effectuer un remodelage de la vacuole. Vps1 est nécessaire pour la fusion et la fragmentation des vacuoles. J'ai découvert que tous les deux processus dépendent de sa capacité d'hydrolyser du GTP. Ainsi l'association avec la membrane est couplée au cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'une autre protéine, et interagit donc très probablement avec les lipides de la membrane. Deux parties différentes de la protéine sont impliquées dans la liaison, dont une, inattendue, le domaine GTPase.¦-¦Numerous organelles undergo membrane fission and fusion events during cell division, vesicular traffic, or in response to changes in environmental conditions. Examples include Golgi (Acharya et al., 1998) mitochondria (Bleazard et al., 1999) peroxisomes (Kuravi et al., 2006) and lysosomes (Ward et al., 1997). In the yeast Saccharomyces cerevisiae the vacuole is the terminal component of the endocytic pathway and corresponds to lysosomes in mammalian cells. Yeast vacuoles fragment into multiple small vesicles in response to a hypertonic shock. This rapid and homogeneous reaction can serve as a model to study the requirements of the fragmentation process. Here, I investigated osmotically induced fragmentation by time-lapse microscopy. I observe that the small fragmentation products originate directly from the large central vacuole by asymmetric scission rather than by consecutive equal divisions and that fragmentation occurs in two distinct phases. During the first minute, vacuoles shrink and generate deep invaginations, leaving behind tubular structures. This phase requires the dynamin-like GTPase Vps1 and the vacuolar proton gradient. In the subsequent 10-15 minutes, vesicles pinch off from the tubular structures in a polarized fashion, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol- 3,5-bisphosphate by the Fab1 complex. I suggest a possible regulation of vacuole fragmentation by the CDK Pho85. Based on my microscopy study I established a sequential involvement of the different fission factors.¦In addition to the morphological description of vacuole fragmentation I more specifically aimed to shed some light on the role of Vps1 in vacuole fragmentation and fusion. I find that both functions are dependent on the GTPase activity of the protein and that also the membrane association of the dynamin-like protein is coupled to the GTPase cycle. I found that Vps1 has the capacity for direct lipid binding on the vacuole and that this lipid binding is at least partially mediated through residues in the GTPase domain, a complete novelty for a dynamin family member. A second stretch located in the region of insert Β has also membrane-binding activity or regulates the association with the vacuole through the GTPase domain. Under the assumption of two membrane-binding regions I speculate on Vps1 as a possible tethering factor for vacuole fusion.

Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

In this study we have examined certain aspects of the process of cell invasion and parasitophorous vacuole escape by metacyclic trypomastigotes and extracellular amastigote forms of Trypanosoma cruzi (G strain). Using Vero (and HeLa) cells as targets, we detected differences in the kinetics of vacuole escape by the two forms. Alcalinization of intercellular pH influenced both invasion as well as the escape from the parasitophorous vacuole by metacyclic trypomastigotes, but not the escape kinetics of extracellular amastigotes. We used sialic acid mutants as target cells and observed that the deficiency of this molecule facilitated the escape of both infective forms. Hemolysin activity was only detected in extracellular amastigotes and neither form presented detectable transialidase activity. Invasion of extracellular amastigotes and trypomastigotes in Vero cells was affected in different ways by drugs that interfere with host cell Ca2+ mobilization. These results are in line with previous results that indicate that metacyclic trypomastigotes and extracellular amastigote forms utilize mechanisms with particular features to invade host cells and to escape from their parasitophorous vacuoles.

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1 Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1 cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.

Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae.

The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

The deubiquitinating enzyme AMSH3 is required for intracellular trafficking and vacuole biogenesis in Arabidopsis thaliana.

Ubiquitination, deubiquitination, and the formation of specific ubiquitin chain topologies have been implicated in various cellular processes. Little is known, however, about the role of ubiquitin in the development of cellular organelles. Here, we identify and characterize the deubiquitinating enzyme AMSH3 from Arabidopsis thaliana. AMSH3 hydrolyzes K48- and K63-linked ubiquitin chains in vitro and accumulates both ubiquitin chain types in vivo. amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. Furthermore, AMSH3 is required for efficient endocytosis of the styryl dye FM4-64 and the auxin efflux facilitator PIN2. We thus present evidence for a role of deubiquitination in intracellular trafficking and vacuole biogenesis.

Leishmania major promastigote entry of an autophagy-like compartment and amastigote escape from the parasitophorous vacuole

In this thesis, we investigated the interaction of the obligate intracellular parasite Leishmania (L.) major with two phenotypes of human monocyte derived macrophages (hMDMs). Thereby we focused on the development and maturation of the parasitophorous vacuole (PV) and could show that compartment development is dependent on the parasite stage.rnFocusing on the ultrastructure of PVs containing axenic amastigotes, we demonstrated that the parasites are partially located in damaged PVs or in the cytoplasm of the host. Moreover, we visualized multiple amastigotes in a common PV 144 h p.i. in pro-inflammatory hMDM I but not in anti-inflammatory hMDM II indicating different PV development. rnRegarding the promastigote form, we demonstrated a different uptake of viable and apoptotic L. major promastigotes by hMDMs. Viable promastigotes are predominantly taken up via the flagellum tip whereas apoptotic promastigotes enter the cells via the parasite body. Analyzing compartment maturation, we found that 20-30% of the PVs get positive for the early maturation markers PI3P and EEA1 independent of the viability of the parasites and unaffected by the human macrophage type. Subsequently, 25-40% of the parasites acquire the autophagy marker LC3 on their PV, what is independent of the viability of the parasites as well. We quantified this and in hMDM II less LC3-positive compartments formed compared to hMDM I. Analyzing the ultrastructure, we investigated that the compartments consist of a single-membrane PV characteristic for LC3-associated phagocytosis (LAP). Involvement of LAP was confirmed by demonstrating that the protein kinase ULK1 is dispensable for LC3-compartment formation around Leishmania PVs. Visualizing compartment dynamics in real time showed that apoptotic promastigotes are degraded in LC3-positve compartments, whereas viable promastigotes are able to get rid of LC3-protein on their PV suggesting an involvement in parasite development and survival. In this thesis, we established a lentiviral based fluorescent imaging technique that we combined with High-Pressure-Freezing (HPF) and high-resolution 3D electron microscopy. We visualized a promastigote in a LC3-compartment whose ultrastructure showed an opening of the PV to the outside. To identify new LAP markers involved in Leishmania infection, we established an immuno-magnetic isolation protocol for the purification of Leishmania containing compartments.rnIn conclusion, this study suggests that L. major compartment biogenesis and maturation in pro- and anti-inflammatory human macrophages is dependent on the parasite stage and is different between axenic amastigotes, viable promastigotes and apoptotic promastigotes. Understanding the development and maturation of Leishmania parasites in human host cells is important to control and combat the neglected disease leishmaniasis in the future.rn

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

A Plasmodium phospholipase is involved in disruption of the liver stage parasitophorous vacuole membrane

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.

δ-Tonoplast intrinsic protein defines unique plant vacuole functions

Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), δ-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of δ-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity.

The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth

The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the polarized movement of several organelles and is essential for yeast viability. We have shown previously that Myo2p is required for the movement of a portion of the lysosome (vacuole) into the bud and consequently for proper inheritance of this organelle during cell division. Class V myosins have a globular carboxyl terminal tail domain that is proposed to mediate localization of the myosin, possibly through interaction with organelle-specific receptors. Here we describe a myo2 allele whose phenotypes support this hypothesis. vac15–1/myo2–2 has a single mutation in this globular tail domain, causing defects in vacuole movement and inheritance. Although a portion of wild-type Myo2p fractionates with the vacuole, the myo2–2 gene product does not. In addition, the mutant protein does not concentrate at sites of active growth, the predominant location of wild-type Myo2p. Although deletion of the tail domain is lethal, the myo2–2 gene product retains the essential functions of Myo2p. Moreover, myo2–2 does not cause the growth defects and lethal genetic interactions seen in myo2–66, a mutant defective in the actin-binding domain. These observations suggest that the myo2–2 mutation specifically disrupts interactions with selected myosin receptors, namely those on the vacuole membrane and those at sites of polarized growth.