Allophagy

In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.     

Spermidine: A novel autophagy inducer and longevity elixir

Spermidine is a ubiquitous polycation that is synthesized from putrescine and serves as a precursor of spermine. Putrescine, spermidine and spermine all are polyamines that participate in multiple known and unknown biological processes. Exogenous supply of spermidine prolongs the life span of several model organisms including yeast (Saccharomyces cerevisiae), nematodes (Caenorhabditis elegans) and flies (Drosophila melanogaster) and significantly reduces age-related oxidative protein damage in mice, indicating that this agent may act as a universal anti-aging drug. Spermidine induces autophagy in cultured yeast and mammalian cells, as well as in nematodes and flies. Genetic inactivation of genes essential for autophagy abolishes the life span-prolonging effect of spermidine in yeast, nematodes and flies. These findings complement expanding evidence that autophagy mediates cytoprotection against a variety of noxious agents and can confer longevity when induced at the whole-organism level. We hypothesize that increased autophagic turnover of cytoplasmic organelles or long-lived proteins is involved in most if not all life span-prolonging therapies.     

The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast

The budding yeast shmoo tip is a model system for analyzing mechanisms coupling force production to microtubule plus-end polymerization/depolymerization. Dynamic plus ends of astral microtubules interact with the shmoo tip in mating yeast cells, positioning nuclei for karyogamy. We have used live-cell imaging of GFP fusions to identify proteins that couple dynamic microtubule plus ends to the shmoo tip. We find that Kar3p, a minus end-directed kinesin motor protein, is required, whereas the other cytoplasmic motors, dynein and the kinesins Kip2p and Kip3p, are not. In the absence of Kar3p, attached microtubule plus ends released from the shmoo tip when they switched to depolymerization. Furthermore, microtubules in cells expressing kar3-1, a mutant that results in rigor binding to microtubules [2], were stabilized specifically at shmoo tips. Imaging of Kar3p-GFP during mating revealed that fluorescence at the shmoo tip increased during periods of microtubule depolymerization. These data are the first to localize the activity of a minus end-directed kinesin at the plus ends of microtubules. We propose a model in which Kar3p couples depolymerizing microtubule plus ends to the cell cortex and the Bim1p-Kar9p protein complex maintains attachment during microtubule polymerization. In support of this model, analysis of Bim1p-GFP at the shmoo tip results in a localization pattern complementary to that of Kar3p-GFP.     

Discovery of pan autophagy inhibitors through a high-throughput screen highlights macroautophagy as an evolutionarily conserved process across 3 eukaryotic kingdoms

Due to the involvement of macroautophagy/autophagy in different pathophysiological conditions such as infections, neurodegeneration and cancer, identification of novel small molecules that modulate the process is of current research and clinical interest. In this work, we developed a luciferase-based sensitive and robust kinetic high-throughput screen (HTS) of small molecules that modulate autophagic degradation of peroxisomes in the budding yeast Saccharomyces cerevisiae. Being a pathway-specific rather than a target-driven assay, we identified small molecule modulators that acted at key steps of autophagic flux. Two of the inhibitors, Bay11 and ZPCK, obtained from the screen were further characterized using secondary assays in yeast. Bay11 inhibited autophagy at a step before fusion with the vacuole whereas ZPCK inhibited the cargo degradation inside the vacuole. Furthermore, we demonstrated that these molecules altered the process of autophagy in mammalian cells as well. Strikingly, these molecules also modulated autophagic flux in a novel model plant, Aponogeton madagascariensis. Thus, using small molecule modulators identified by using a newly developed HTS autophagy assay, our results support that macroautophagy is a conserved process across fungal, animal and plant kingdoms.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.     

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.     

Longevity-relevant regulation of autophagy at the level of the acetylproteome

The acetylase inhibitor, spermidine and the deacetylase activator, resveratrol, both induce autophagy and prolong life span of the model organism Caenorhabditis elegans in an autophagydependent fashion. Based on these premises, we investigated the differences and similarities in spermidine and resveratrol-induced autophagy. The deacetylase sirtuin 1 (SIRT1) and its orthologs are required for the autophagy induction by resveratrol but dispensable for autophagy stimulation by spermidine in human cells, Saccharomyces cerevisiae and C. elegans. SIRT1 is also dispensable for life-span extension by spermidine. Mass spectrometry analysis of the human acetylproteome revealed that resveratrol and/or spermidine induce changes in the acetylation of 560 peptides corresponding to 375 different proteins. Among these, 170 proteins are part of the recently elucidated human autophagy protein network. Importantly, spermidine and resveratrol frequently affect the acetylation pattern in a similar fashion. In the cytoplasm, spermidine and resveratrol induce convergent protein de-acetylation more frequently than convergent acetylation, while in the nucleus, acetylation is dominantly triggered by both agents. We surmise that subtle and concerted alterations in the acetylproteome regulate autophagy at multiple levels.     

A link between very long chain fatty acid elongation and mating-specific yeast cell cycle arrest

Ceramides and sphingolipid intermediates are well-established regulators of the cell cycle. In the budding yeast Saccharomyces cerevisae, the complex sphingolipid backbone, ceramide, comprises a long chain sphingoid base, a polar head group, and a very long chain fatty acid (VLCFA). While ceramides and long chain bases have been extensively studied as to their roles in regulating cell cycle arrest under multiple conditions, the roles of VLCFAs are not well understood. Here, we used the yeast elo2 and elo3 mutants, which are unable to elongate fatty acids, as tools to explore if maintaining VLCFA elongation is necessary for cell cycle arrest in response to yeast mating. We found that both elo2 and elo3 cells had severely reduced mating efficiencies and were unable to form polarized shmoo projections that are necessary for cell-cell contact during mating. They also lacked functional MAP kinase signaling activity and were defective in initiating a cell cycle arrest in response to pheromone. Additional data suggests that mislocalization of the Ste5 scaffold in elo2 and elo3 mutants upon mating initiation may be responsible for the inability to initiate a cell cycle arrest. Moreover, the lack of proper Ste5 localization may be caused by the inability of mutant cells to mobilize PIP₂. We suggest that VLCFAs are required for Ste5 localization, which is a necessary event for initiating MAP kinase signaling and cell cycle arrest during yeast mating initiation.     

Spermidine-triggered autophagy ameliorates memory during aging

The aging process drives the progressive deterioration of an organism and is thus subject to a complex interplay of regulatory and executing mechanisms. Our understanding of this process eventually aims at the delay and/or prevention of age-related pathologies, among them the age-dependent decrease in cognitive performance (e.g., learning and memory). Using the fruit fly Drosophila melanogaster, which combines a generally high mechanistic conservation with an efficient experimental access regarding aging and memory studies, we have recently unveiled a protective function of polyamines (including spermidine) against age-induced memory impairment (AMI). The flies’ age-dependent decline of aversive olfactory memory, an established model for AMI, can be rescued by both pharmacological treatment with spermidine and genetic modulation that increases endogenous polyamine levels. Notably, we find that this effect strictly depends on autophagy, which is remarkable in light of the fact that autophagy is considered a key regulator of aging in other contexts. Given that polyamines in general and spermidine in particular are endogenous metabolites, our findings place them as candidate target substances for AMI treatment.     

Prm1 Targeting to Contact Sites Enhances Fusion during Mating in Saccharomyces cerevisiae

Prm1 is a pheromone-regulated membrane glycoprotein involved in the plasma membrane fusion event of Saccharomyces cerevisiae mating. Although this function suggests that Prm1 should act at contact sites in pairs of mating yeast cells where plasma membrane fusion occurs, only a small percentage of the total Prm1 was actually detected on the plasma membrane. We therefore investigated the intracellular transport of Prm1 and how this transport contributes to cell fusion. Two Prm1 chimeras that were sorted away from the contact site had reduced fusion activity, indicating that Prm1 indeed functions at contact sites. However, most Prm1 is located in endosomes and other cytoplasmic organelles and is targeted to vacuoles for degradation. Mutations in a putative endocytosis signal in a cytoplasmic loop partially stabilized the Prm1 protein and caused it to accumulate on the plasma membrane, but this endocytosis mutant actually had reduced mating activity. When Prm1 was expressed from a galactose-regulated promoter and its synthesis was repressed at the start of mating, vanishingly small amounts of Prm1 protein remained at the time when the plasma membranes came into contact. Nevertheless, this stable pool of Prm1 was retained at polarized sites on the plasma membrane and was sufficient to promote plasma membrane fusion. Thus, the amount of Prm1 expressed in mating yeast is far in excess of the amount required to facilitate fusion.Membrane fusion has been studied extensively in the context of viral infection and intracellular membrane fusion. These fusion events are mediated by fusases—proteins that mediate membrane fusion. Some of the best-studied fusases are the SNAREs (soluble N-ethylmaleimide-sensitive factors) that mediate fusion of intracellular organelles and the hemagglutinin (HA) protein of influenza virus that mediates fusion of the viral envelope membrane with host endosomes (13). However, little is known about how the plasma membranes of two cells fuse during cell fusion.Cell fusion is essential for the development of multicellular organisms. Some cell fusion processes involve a single pair of cells, as in sperm-egg fusion. Many other developmental processes require multiple fusion events, as in fusion of myoblasts for muscle formation. However, all fusion events must overcome a common obstacle—maintaining the integrity and selective permeability of the two plasma membranes while fusing the hydrophobic cores of their phospholipid bilayers.We study cell fusion in mating pairs of the yeast Saccharomyces cerevisiae. This organism offers a genetically tractable model amenable to many biochemical and cell biological assays. The mating pathway in yeast is comprised of 5 steps: pheromone signaling, adhesion, degradation of the intervening cell walls, plasma membrane fusion, and karyogamy. S. cerevisiae has two haploid mating types: MATa and MATα. Haploid cells secrete pheromones that bind to G-protein-coupled receptors on the surface of cells of the opposite mating type. Pheromone binding activates a signaling cascade that causes cell cycle arrest, expression of pheromone-inducible genes, and polarized growth to form a mating projection (or shmoo tip). The binding of two cells of opposite mating type to form a mating pair is mediated by complementary agglutinins located on the shmoo tips. Then, the cell walls of the two cells are joined to form a unified wall protecting the mating pair, and the walls between the two cells are degraded. This allows the plasma membranes to come into contact and fuse. The initial fusion pore between cells expands to allow cytoplasmic mixing and, ultimately, karyogamy. After mating is complete, the mitotic cell cycle resumes, and a diploid daughter cell buds from the neck connecting the two parent cells (5, 30).This work focuses on Prm1, a glycoprotein that promotes the plasma membrane fusion step of mating. PRM1 was discovered in a bioinformatic screen designed to identify Prm (pheromone-regulated membrane) proteins (11). Prm1 has four transmembrane domains and functions as a disulfide-linked dimer (20). Prm1-deficient mating pairs experience one of three fates: arrest as late prezygotes (unfused mating pairs with no intervening cell walls), lysis once their plasma membranes come into contact, or fusion. Electron microscopy revealed that the two plasma membranes in a late prezygote were only ∼8 nm apart but did not fuse. Additional studies showed that ∼30% of prm1Δ mating pairs lyse after membrane contact (1, 14). However, 50% of prm1Δ mating pairs fuse on standard yeast extract-peptone-dextrose (YPD) medium, implying that Prm1 is important, but not required, for fusion. Mating becomes more dependent upon Prm1 activity if Ca²⁺ or ergosterol is limiting (1, 15).On the basis of its apparent role in membrane fusion, Prm1 should be targeted to the contact sites where membranes fuse. Surprisingly, only a small amount of Prm1 was found at contact sites, and even less was at shmoo tips or at bud tips in mitotic cells expressing Prm1 from a constitutive promoter. These observations prompted further investigation of Prm1''s intracellular transport. The results revealed that Prm1 does indeed function at contact sites. However, except for the small pool that promotes fusion, Prm1 proteins are transported to vacuoles and rapidly degraded.     

Spermidine protects against α-synuclein neurotoxicity

As our society ages, neurodegenerative disorders like Parkinson`s disease (PD) are increasing in pandemic proportions. While mechanistic understanding of PD is advancing, a treatment with well tolerable drugs is still elusive. Here, we show that administration of the naturally occurring polyamine spermidine, which declines continuously during aging in various species, alleviates a series of PD-related degenerative processes in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, two established model systems for PD pathology. In the fruit fly, simple feeding with spermidine inhibited loss of climbing activity and early organismal death upon heterologous expression of human α-synuclein, which is thought to be the principal toxic trigger of PD. In this line, administration of spermidine rescued α-synuclein-induced loss of dopaminergic neurons, a hallmark of PD, in nematodes. Alleviation of PD-related neurodegeneration by spermidine was accompanied by induction of autophagy, suggesting that this cytoprotective process may be responsible for the beneficial effects of spermidine administration.     

Pheromone-encoding mRNA is transported to the yeast mating projection by specific RNP granules

Association of messenger RNAs with large complexes such as processing bodies (PBs) plays a pivotal role in regulating their translation and decay. Little is known about other possible functions of these assemblies. Exposure of haploid yeast cells, carrying mating type “a,” to “α pheromone” stimulates polarized growth resulting in a “shmoo” projection; it also induces synthesis of “a pheromone,” encoded by MFA2. In this paper, we show that, in response to α pheromone, MFA2 mRNA is assembled with two types of granules; both contain some canonical PB proteins, yet they differ in size, localization, motility, and sensitivity to cycloheximide. Remarkably, one type is involved in mRNA transport to the tip of the shmoo, whereas the other—in local translation in the shmoo. Normal assembly of these granules is critical for their movement, localization, and for mating. Thus, MFA2 mRNAs are transported to the shmoo tip, in complex with PB-like particles, where they are locally translated.     

Maternal inheritance of mitochondrial DNA

Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.     

Allophagy: A macroautophagic process degrading spermatozoid-inherited organelles

In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.     

A lifespan-extending form of autophagy employs the vacuole-vacuole fusion machinery

While autophagy is believed to be beneficial for lifespan extension, it is controversial which forms or aspects of autophagy are the responsible ones. We addressed this by analyzing the lifespan of yeast autophagy mutants under caloric restriction, a longevity manipulation. Surprisingly, we discovered that the majority of proteins involved in macro-autophagy and several forms of micro-autophagy were dispensable for lifespan extension. The only autophagy protein that is critical for lifespan extension was Atg15p, a lipase that is located in the endoplasmic reticulum (ER) and transported to vacuoles for disintegrating membranes of autophagic bodies. We further found that vacuole-vacuole fusion was required for lifespan extension, which was indicated by the shortened lifespan of mutants missing proteins (ypt7Δ, nyv1Δ, vac8Δ) or lipids (erg6Δ) involved in fusion. Since a known function of vacuole-vacuole fusion is the maintenance of the vacuole membrane integrity, we analyzed aged vacuoles and discovered that aged cells had altered vacuolar morphology and accumulated autophagic bodies, suggesting that certain forms of autophagy do contribute to longevity. Like aged cells, erg6Δ accumulated autophagic bodies, which is likely caused by a defect in lipase instead of proteases due to the existence of multiple vacuolar proteases. Since macro-autophagy is not blocked by erg6Δ, we propose that a new form of autophagy transports Atg15p via the fusion of vacuoles with vesicles derived from ER, and we designate this putative form of autophagy as secretophagy. Pending future biochemical studies, the concept of secretophagy may provide a mechanism for autophagy in lifespan extension.     

Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.     

Overexpression of Autophagy-Related Genes Inhibits Yeast Filamentous Growth

Under conditions of nitrogen stress, the budding yeast S. cerevisiae initiates a cellular response involving the activation of autophagy, an intracellular catabolic process for the degradation and recycling of proteins and organelles. In certain strains of yeast, nitrogen stress also drives a striking developmental transition to a filamentous form of growth, in which cells remain physically connected after cytokinesis. We recently identified an interrelationship between these processes, with the inhibition of autophagy resulting in exaggerated filamentous growth. Our results suggest a model wherein autophagy mitigates nutrient stress, and filamentous growth is responsive to the degree of this stress. Here, we extended these studies to encompass a phenotypic analysis of filamentous growth upon overexpression of autophagy-related (ATG) genes. Specifically, overexpression of ATG1, ATG3, ATG7, ATG17, ATG19, ATG23, ATG24, and ATG29 inhibited filamentous growth. From our understanding of autophagy in yeast, overexpression of these genes does not markedly affect the activity of the pathway; thus, we do not expect that this filamentous growth phenotype is due strictly to diminished nitrogen stress in ATG overexpression mutants. Rather, these results highlight an additional undefined regulatory mechanism linking autophagy and filamentous growth, possibly independent of the upstream nitrogen-sensing machinery feeding into both processes.

Addendum to:

An Interrelationship Between Autophagy and Filamentous Growth in Budding Yeast

J. Ma, R. Jin, X. Jia, C.J. Dobry, L. Wang, F. Reggiori, J. Zhu and A. Kumar

Genetics 2007; In press     

Commentary: Spermidine-triggered autophagy ameliorates memory during aging

The aging process drives the progressive deterioration of an organism and is thus subject to a complex interplay of regulatory and executing mechanisms. Our understanding of this process eventually aims at the delay and/or prevention of age-related pathologies, among them the age-dependent decrease in cognitive performance (e.g., learning and memory). Using the fruit fly Drosophila melanogaster, which combines a generally high mechanistic conservation with an efficient experimental access regarding aging and memory studies, we have recently unveiled a protective function of polyamines (including spermidine) against age-induced memory impairment (AMI). The flies’ age-dependent decline of aversive olfactory memory, an established model for AMI, can be rescued by both pharmacological treatment with spermidine and genetic modulation that increases endogenous polyamine levels. Notably, we find that this effect strictly depends on autophagy, which is remarkable in light of the fact that autophagy is considered a key regulator of aging in other contexts. Given that polyamines in general and spermidine in particular are endogenous metabolites, our findings place them as candidate target substances for AMI treatment.     

The Glc7p-interacting protein Bud14p attenuates polarized growth,pheromone response,and filamentous growth in Saccharomyces cerevisiae

A genetic selection in Saccharomyces cerevisiae for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth. Cloning and segregation analysis demonstrated that BUD14 was the affected gene. Disruption of BUD14 in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology. The bud14 mutant also had hyperfilamentous growth. Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell. Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p. The genetic interactions between BUD14 and GLC7 suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast.