The role of Myo2, a yeast class V myosin,in vesicular transport

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post- Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy- peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.     

The peroxisomal membrane protein Inp2p is the peroxisome-specific receptor for the myosin V motor Myo2p of Saccharomyces cerevisiae

The faithful inheritance of organelles by daughter cells is essential to maintain the benefits afforded to eukaryotic cells by compartmentalization of biochemical functions. In Saccharomyces cerevisiae, the class V myosin, Myo2p, is involved in transporting different organelles, including the peroxisome, along actin cables to the bud. We identified Inp2p as the peroxisome-specific receptor for Myo2p. Cells lacking Inp2p fail to partition peroxisomes to the bud but are unaffected in the inheritance of other organelles. Inp2p is a peroxisomal membrane protein, preferentially enriched in peroxisomes delivered to the bud. Inp2p interacts directly with the globular tail of Myo2p. Cells overproducing Inp2p often transfer their entire populations of peroxisomes to buds. The levels of Inp2p oscillate with the cell cycle. Organelle-specific receptors like Inp2p explain how a single motor can move different organelles in distinct and specific patterns. To our knowledge, Inp2p is the first peroxisomal protein implicated in the vectorial movement of peroxisomes.     

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 unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae

Myo2p is an unconventional myosin required for polarized growth in Saccharomyces cerevisiae. Four lines of evidence suggest that (a) Myo2p is a target of calmodulin at sites of cell growth, and (b) the interaction between Myo2p and calmodulin is Ca2+ independent. First, as assessed by indirect immunofluorescence, the distributions of Myo2p and calmodulin are nearly indistinguishable throughout the cell cycle. Second, a genetic analysis indicates that mutations in CMD1 show allele- specific synthetic lethality with the myo2-66 conditional mutation. Mutations that inactivate the Ca(2+)-binding sites of calmodulin have little or no effect on strains carrying myo2-66, whereas an allele with a mutation outside the Ca(2+)-binding sites dramatically increases the severity of the phenotype conferred by myo2-66. Third, Myo2p coimmunoprecipitates with calmodulin in the presence of Ca2+ or EGTA. Finally, we used a modified gel overlay assay to demonstrate direct interaction between calmodulin and fusion proteins containing portions of Myo2p. Calmodulin binds specifically to the region of Myo2p containing six tandem repeats of a motif called an IQ site. Binding occurs in either Ca2+ or EGTA, and only two sites are required to observe binding.     

The Sla2p talin domain plays a role in endocytosis in Saccharomyces cerevisiae

Clathrin-binding adaptors play critical roles for endocytosis in multicellular organisms, but their roles in budding yeast have remained unclear. To address this question, we created a quadruple mutant yeast strain lacking the genes encoding the candidate clathrin adaptors Yap1801p, Yap1802p, and Ent2p and containing a truncated version of Ent1p, Ent1DeltaCBMp, missing its clathrin-binding motif. This strain was viable and competent for endocytosis, suggesting the existence of other redundant adaptor-like factors. To identify these factors, we mutagenized the quadruple clathrin adaptor mutant strain and selected cells that were viable in the presence of full-length Ent1p, but inviable with only Ent1DeltaCBMp; these strains were named Rcb (requires clathrin binding). One mutant strain, rcb432, contained a mutation in SLA2 that resulted in lower levels of a truncated protein lacking the F-actin binding talin homology domain. Analyses of this sla2 mutant showed that the talin homology domain is required for endocytosis at elevated temperature, that SLA2 exhibits genetic interactions with both ENT1 and ENT2, and that the clathrin adaptors and Sla2p together regulate the actin cytoskeleton and revealed conditions under which Yap1801p and Yap1802p contribute to viability. Together, our data support the view that Sla2p is an adaptor that links actin to clathrin and endocytosis.     

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.     

Characterization of Mmp37p, a Saccharomyces cerevisiae mitochondrial matrix protein with a role in mitochondrial protein import

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.     

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.     

The MAP kinase-activated protein kinase Rck2p plays a role in rapamycin sensitivity in Saccharomyces cerevisiae and Candida albicans

Rck2p is a Hog1p-MAP kinase-activated protein kinase and regulates osmotic and oxidative stresses in budding yeast. In this study, we have demonstrated in both Saccharomyces cerevisiae and, the most medically important human fungal pathogen, Candida albicans that deletion of RCK2 renders cells sensitive to rapamycin, the inhibitor of target of rapamycin protein kinase controlling cell growth. The kinase activity of Rck2p does not seem to be required for this rapamycin sensitivity function in both eukaryotic microorganisms. Interestingly, the HOG pathway is not directly involved in cell sensitivity to rapamycin in S. cerevisiae, whereas disruption of CaHOG1 renders cells sensitive to rapamycin in C. albicans. In addition, we have shown that CaRck2p and its kinase activity are required for cell growth in C. albicans.     

Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae

Summary The nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA. Genetic studies have shown that the target sequence of the CBS2 protein is the 5 untranslated leader sequence of cytochrome b RNA. Here we report on the intracellular localization of CBS2. CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum. Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted. The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant. Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro. Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.     

A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae.

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Delta mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Delta cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.     

GTP drives myosin light chain 1 interaction with the class V myosin Myo2 IQ motifs via a Sec2 RabGEF-mediated pathway

The yeast myosin light chain 1 (Mlc1p) belongs to a branch of the calmodulin superfamily and is essential for vesicle delivery at the mother-bud neck during cytokinesis due to is ability to bind to the IQ motifs of the class V myosin Myo2p. While calcium binding to calmodulin promotes binding/release from the MyoV IQ motifs, Mlc1p is unable to bind calcium and the mechanism of its interaction with target motifs has not been clarified. The presence of Mlc1p in a complex with the Rab/Ypt Sec4p and with Myo2p suggests a role for Mlc1p in regulating Myo2p cargo binding/release by responding to the activation of Rab/Ypt proteins. Here we show that GTP or GTPgammaS potently stimulate Mlc1p interaction with Myo2p IQ motifs. The C-terminus of the Rab/Ypt GEF Sec2p, but not Sec4p activation, is essential for this interaction. Interestingly, overexpression of constitutively activated Ypt32p, a Rab/Ypt protein that acts upstream of Sec4p, stimulates Mlc1p/Myo2p interaction similarly to GTP although a block of Ypt32 GTP binding does not completely abolish the GTP-mediated Mlc1p/Myo2p interaction. We propose that Mlc1p/Myo2p interaction is stimulated by a signal that requires Sec2p and activation of Ypt32p.     

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.     

The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.     

The myosin-related motor protein Myo2 is an essential mediator of bud-directed mitochondrial movement in yeast

The inheritance of mitochondria in yeast depends on bud-directed transport along actin filaments. It is a matter of debate whether anterograde mitochondrial movement is mediated by the myosin-related motor protein Myo2 or by motor-independent mechanisms. We show that mutations in the Myo2 cargo binding domain impair entry of mitochondria into the bud and are synthetically lethal with deletion of the YPT11 gene encoding a rab-type guanosine triphosphatase. Mitochondrial distribution defects and synthetic lethality were rescued by a mitochondria-specific Myo2 variant that carries a mitochondrial outer membrane anchor. Furthermore, immunoelectron microscopy revealed Myo2 on isolated mitochondria. Thus, Myo2 is an essential and direct mediator of bud-directed mitochondrial movement in yeast. Accumulating genetic evidence suggests that maintenance of mitochondrial morphology, Ypt11, and retention of mitochondria in the bud contribute to Myo2-dependent inheritance of mitochondria.     

Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5 cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and of grx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.     

The C-terminal extension of Saccharomyces cerevisiae Hsp104 plays a role in oligomer assembly

The Saccharomyces cerevisiae protein Hsp104, a member of the Hsp100/Clp AAA+ family of ATPases, and its orthologues in plants (Hsp101) and bacteria (ClpB) function to disaggregate and refold thermally denatured proteins following heat shock and play important roles in thermotolerance. The primary sequences of fungal Hsp104's contain a largely acidic C-terminal extension not present in bacterial ClpB's. In this work, deletion mutants were used to determine the role this extension plays in Hsp104 structure and function. Elimination of the C-terminal tetrapeptide DDLD diminishes binding of the tetratricopeptide repeat domain cochaperone Cpr7 but is dispensable for Hsp104-mediated thermotolerance. The acidic region of the extension is also dispensable for thermotolerance and for the stimulation of Hsp104 ATPase activity by poly-l-lysine, but its truncation results in an oligomerization defect and reduced ATPase activity in vitro. Finally, sequence alignments reveal that the C-terminal extension contains a sequence (VLPNH) that is conserved in fungal Hsp104's but not in other orthologues. Hsp104 lacking the entire C-terminal extension including the VLPNH region does not assemble and has very low ATPase activity. In the presence of a molecular crowding agent the ATPase activities of mutants with longer truncations are partially restored possibly through enhanced oligomer formation. However, elimination of the whole C-terminal extension results in an Hsp104 molecule which is unable to assemble and becomes aggregation prone at high temperature, highlighting a novel structural role for this region.