Activation of the Mitogen-activated Protein Kinase,Slt2p,at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae. Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.     

A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer of information and material between both organelles. During budding, directed movement of cER to the young bud followed by subsequent anchorage at its tip ensures the faithful inheritance of this organelle. The ER membrane protein Scs2p tethers the cER to the PM and to the bud tip through so far unknown receptors. We characterize Epo1p as a novel member of the polarisome that interacts with Scs2p exclusively at the cell tip during bud growth and show that Epo1p binds simultaneously to the Cdc42p guanosine triphosphatase–activating protein Bem3p. Deletion of EPO1 or deletion of BEM3 in a polarisome-deficient strain reduces the amount of cER at the tip. This analysis therefore identifies Epo1p as a novel and important component of the polarisome that promotes cER tethering at sites of polarized growth.     

Ptc1p regulates cortical ER inheritance via Slt2p

Studies in the yeast Saccharomyces cerevisiae have shown that the inheritance of endoplasmic reticulum (ER), mitochondria, and vacuoles involves the capture of a tubular structure at the bud tip. Ptc1p, a serine/threonine phosphatase, has previously been shown to regulate mitochondrial inheritance by an unknown mechanism. Ptc1p regulates the high osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway and has also been implicated in the cell wall integrity (CWI) MAPK pathway. Here we show that the loss of Ptc1p or the Ptc1p binding protein, Nbp2p, causes a prominent delay in the delivery of ER tubules to the periphery of daughter cells and results in a dramatic increase in the level of phosphorylated Slt2p, the MAPK in the CWI pathway. Either loss of Slt2p or inhibition of the CWI pathway by addition of sorbitol, suppresses the ER inheritance defect in the ptc1Delta and nbp2Delta mutants. Our findings indicate that Ptc1p and Nbp2p regulate ER inheritance through the CWI MAPK pathway by modulating the MAPK, Slt2p.     

Mitochondrial Inheritance Is Delayed in Saccharomyces cerevisiae Cells Lacking the Serine/Threonine Phosphatase PTC1

In wild-type yeast mitochondrial inheritance occurs early in the cell cycle concomitant with bud emergence. Cells lacking the PTC1 gene initially produce buds without a mitochondrial compartment; however, these buds later receive part of the mitochondrial network from the mother cell. Thus, the loss of PTC1 causes a delay, but not a complete block, in mitochondrial transport. PTC1 encodes a serine/threonine phosphatase in the high-osmolarity glycerol response (HOG) pathway. The mitochondrial inheritance delay in the ptc1 mutant is not attributable to changes in intracellular glycerol concentrations or defects in the organization of the actin cytoskeleton. Moreover, epistasis experiments with ptc1Δ and mutations in HOG pathway kinases reveal that PTC1 is not acting through the HOG pathway to control the timing of mitochondrial inheritance. Instead, PTC1 may be acting either directly or through a different signaling pathway to affect the mitochondrial transport machinery in the cell. These studies indicate that the timing of mitochondrial transport in wild-type cells is genetically controlled and provide new evidence that mitochondrial inheritance does not depend on a physical link between the mitochondrial network and the incipient bud site.     

Role for cER and Mmr1p in anchorage of mitochondria at sites of polarized surface growth in budding yeast

Mitochondria accumulate at neuronal and immunological synapses and yeast bud tips and associate with the ER during phospholipid biosynthesis, calcium homeostasis, and mitochondrial fission. Here we show that mitochondria are associated with cortical ER (cER) sheets underlying the plasma membrane in the bud tip and confirm that a deletion in YPT11, which inhibits cER accumulation in the bud tip, also inhibits bud tip anchorage of mitochondria. Time-lapse imaging reveals that mitochondria are anchored at specific sites in the bud tip. Mmr1p, a member of the DSL1 family of tethering proteins, localizes to punctate structures on opposing surfaces of mitochondria and cER sheets underlying the bud tip and is recovered with isolated mitochondria and ER. Deletion of MMR1 impairs bud tip anchorage of mitochondria without affecting mitochondrial velocity or cER distribution. Deletion of the phosphatase PTC1 results in increased Mmr1p phosphorylation, mislocalization of Mmr1p, defects in association of Mmr1p with mitochondria and ER, and defects in bud tip anchorage of mitochondria. These findings indicate that Mmr1p contributes to mitochondrial inheritance as a mediator of anchorage of mitochondria to cER sheets in the yeast bud tip and that Ptc1p regulates Mmr1p phosphorylation, localization, and function.     

Rvs161p and sphingolipids are required for actin repolarization following salt stress

In Saccharomyces cerevisiae, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 min, and then actin patches repolarized. Rvs161p was required for actin repolarization because the rvs161Δ mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the rvs161Δ-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis: FEN1, SUR4, SUR2, SUR1, and IPT1. These suppressors also suppressed act1-1-related salt sensitivity and the defect in actin repolarization of the rvs161Δ mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the rvs161Δ defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs161p in the other suppressor mutants. Rvs161p was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvs161p was required for actin repolarization and was found to be located in lipid rafts.     

Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ, pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.     

Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.     

The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.     

Requirement of Phosphoinositides Containing Stearic Acid To Control Cell Polarity

Phosphoinositides (PIPs) are present in very small amounts but are essential for cell signaling, morphogenesis, and polarity. By mass spectrometry, we demonstrated that some PIPs with stearic acyl chains were strongly disturbed in a psi1Δ Saccharomyces cerevisiae yeast strain deficient in the specific incorporation of a stearoyl chain at the sn-1 position of phosphatidylinositol. The absence of PIPs containing stearic acid induced disturbances in intracellular trafficking, although the total amount of PIPs was not diminished. Changes in PIPs also induced alterations in the budding pattern and defects in actin cytoskeleton organization (cables and patches). Moreover, when the PSI1 gene was impaired, a high proportion of cells with bipolar cortical actin patches that occurred concomitantly with the bipolar localization of Cdc42p was specifically found among diploid cells. This bipolar cortical actin phenotype, never previously described, was also detected in a bud9Δ/bud9Δ strain. Very interestingly, overexpression of PSI1 reversed this phenotype.     

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.     

Transport of Axl2p Depends on Erv14p,an ER–Vesicle Protein Related to the Drosophila cornichon Gene Product

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER–vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Δ strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Δ yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.     

Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast

kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9Δ dhc1Δ, kar9Δ jnm1Δ, and kar9Δ act5Δ double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.     

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

Background

Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus.

Methodology/Principal Findings

Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Δ mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Δ cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Δ cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Δ mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p.

Conclusions/Significance

The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Δ cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Δ cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Δ cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target.     

Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.     

PTC1 Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

Organelle inheritance occurs during cell division. In Saccharomyces cerevisiae, inheritance of the vacuole, and the distribution of mitochondria and cortical endoplasmic reticulum are regulated by Ptc1p, a type 2C protein phosphatase. Here we show that PTC1/VAC10 controls the distribution of additional cargoes moved by a myosin-V motor. These include peroxisomes, secretory vesicles, cargoes of Myo2p, and ASH1 mRNA, a cargo of Myo4p. We find that Ptc1p is required for the proper distribution of both Myo2p and Myo4p. Surprisingly, PTC1 is also required to maintain the steady-state levels of organelle-specific receptors, including Vac17p, Inp2p, and Mmr1p, which attach Myo2p to the vacuole, peroxisomes, and mitochondria, respectively. Furthermore, Vac17p fused to the cargo-binding domain of Myo2p suppressed the vacuole inheritance defect in ptc1Δ cells. These findings suggest that PTC1 promotes the association of myosin-V with its organelle-specific adaptor proteins. Moreover, these observations suggest that despite the existence of organelle-specific receptors, there is a higher order regulation that coordinates the movement of diverse cellular components.     

Localization of a Subset of Yeast mRNAs Depends on Inheritance of Endoplasmic Reticulum

Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1‐3 protein machinery in Saccharomyces cerevisiae localizes >30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co‐localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution. By investigating localization of MS2‐tagged mRNAs in yeast defective at various stages of cER segregation, we demonstrate that proper cER segregation is required for localization of only a subset of mRNAs. These mRNAs include WSC2, IST2, EAR1 and SRL1 that encode membrane or ER associated proteins and are expressed during S and G2 phases of the cell cycle when tubular ER movement into the bud occurs. Translation of WSC2 is not required for localization, ruling out co‐translational targeting of this mRNA. Localization of ASH1 mRNA is independent of cER segregation, which is consistent with the expression pattern of ASH1 at late mitosis. Our findings indicate the presence of two different pathways to localize mRNAs to the yeast bud.     

Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins,Mmm1p and Mdm10p

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.Mitochondria are indispensable organelles for normal eukaryotic cell function. Since mitochondria cannot be synthesized de novo, these organelles are inherited, i.e., transferred from mother to daughter during cell division. In the yeast Saccharomyces cerevisiae, vegetative cell division occurs by budding, a form of proliferation in which growth is directed toward the developing bud. Previous studies indicate that mitochondria undergo a series of cell cycle–linked motility events during normal inheritance in yeast (Simon et al., 1997). These are: (a) polarization of mitochondria towards the site of bud emergence in G₁ phase; (b) linear, polarized movement of mitochondria from mother cells to developing buds in S phase; (c) immobilization of newly inherited mitochondria in the bud tip during S and G₂ phases; and (d) release of immobilized mitochondria from the bud tip during M phase.There is mounting evidence that the actin cytoskeleton controls mitochondrial morphology and inheritance during vegetative yeast cell growth. The two major actin structures of yeast observed by light microscopy are patches and cables. Actin cables are bundles of actin filaments that extend from the mother into the bud. Mitochondria colocalize with these actin cables (Drubin et al., 1993; Lazzarino et al., 1994). Moreover, mutations such as deletion of the tropomyosin I gene, TPM1, or the mitochondrial distribution and morphology gene, MDM20, which selectively destabilize actin cables, result in the loss of polarized mitochondrial movement and reduce transfer of mitochondria into buds (Herman et al., 1997; Simon et al., 1997). Together, these studies indicate that normal mitochondrial inheritance in yeast requires association of mitochondria with actin cables.Cell-free studies reveal a possible mechanism underlying actin control of mitochondrial inheritance. Sedimentation assays document binding of mitochondria to the lateral surface of F-actin. This mitochondrial actin-binding activity is ATP-sensitive, saturable, reversible, and mediated by protein(s) on the mitochondrial surface (Lazzarino et al., 1994). In addition, ATP-driven, actin-dependent motor activity has been identified on the surface of mitochondria (Simon et al., 1995). These observations support a model of mitochondrial inheritance whereby mitochondria use an actin-dependent motor to drive their movement from mother to daughter cells along actin cable tracks.Yeast genetic screens have revealed several genes, collectively referred to as mdm (mitochondrial distribution and morphology) and mmm (maintenance of mitochondrial morphology), which are required for mitochondrial inheritance (McConnell et al., 1990; Burgess et al., 1994; Sogo and Yaffe, 1994). We have focused on two of these genes: MDM10 and MMM1. Deletion of MDM10 leads to the development of giant spherical mitochondria, presumably by the collapse of elongated mitochondria into a spherical mass (Sogo and Yaffe, 1994). Deletion of MMM1 (Burgess et al., 1994) produces a similar phenotype. In both mutants, the fraction of buds without mitochondria is high, indicating defective mitochondrial inheritance. The proteins encoded by these genes, Mdm10p and Mmm1p, appear to be integral membrane proteins in the mitochondrial outer membrane. Here, we report tests of the hypothesis that Mmm1p and Mdm10p are required to link mitochondria to the cytoskeleton.     

Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.     

The SLT2 (MPK1) MAP kinase homolog is involved in polarized cell growth in Saccharomyces cerevisiae

Bud emergence, spindle pole body duplication and DNA replication are all dependent on the activation of the CDC28 protein kinase at the Start point in the G1 phase of the cell cycle. Bud emergence requires polarization of the cytoskeleton and secretory vesicles to a specific site on the cell surface. Cdc28p activated by G1-cyclins triggers polarization of actin to the site of bud emergence and favors apical bud growth (Lew, D. J., and S. I. Reed. 1993. J. Cell Biol. 120:1305- 1320). We isolated slt2-1 as a mutation that enhances the division defect of cdc28 mutants with defects at Start. Slt2p(Mpk1p) is a member of the MAP kinase family (Lee, K. S., K. Irie, Y. Gotoh, Y. Watanabe, H. Araki, E. Nishida, K. Matsumoto, and D. E. Levin. 1993. Mol. Cell. Biol. 13:3067-3075). We show that slt2 mutants exhibit phenotypes similar to those shown by mutants of the yeast actin cytoskeleton, including delocalization of chitin deposition and of actin cortical spots and the accumulation of secretory pathway membranes and vesicles. Furthermore, slt2::HIS3 act1-1 and slt2::HIS3 myo2-66 double mutants are inviable. We suggest that Slt2p functions downstream or in parallel with Cdc28p in promoting bud formation and apical growth.