Shmoo Tip Research Articles

Polarization and cell-fate decision facilitated by the adaptor Ste50p in Saccharomyces cerevisiae.

In response to pheromone, many proteins localize on the plasma membrane of yeast cell to reform it into a polarized shmoo structure. The adaptor protein Ste50p, known as a pheromone signal enhancer critical for shmoo polarization, has never been explored systematically for its localization and function in the polarization process. Time-lapse single-cell imaging and quantitation shown here characterizes Ste50p involvement in the establishment of cell polarity. We found that Ste50p patches on the cell cortex mark the point of shmoo initiation, these patches could move, and remain associated with the growing shmoo tip in a pheromone concentration time-dependent manner until shmoo maturation. A Ste50p mutant impaired in patch localization suffers a delay in polarization. By quantitative analysis we show that polarization correlates with the rising levels of Ste50p, enabling rapid cell responses to pheromone that correspond to a critical level of Ste50p at the initial G1 phase. We exploited the quantitative differences in the pattern of Ste50p expression to correlate with the cell-cell phenotypic heterogeneity, showing Ste50p involvement in the cellular differentiation choice. Taken together, these findings present Ste50p to be part of the early shmoo development phase, suggesting that Ste50p may be involved with the polarisome in the initiation of polarization, and plays a role in regulating the polarized growth of shmoo during pheromone response.

Two- and Three-Dimensional Tracking of MFA2 mRNA Molecules in Mating Yeast.

Intracellular mRNA transport contributes to the spatio-temporal regulation of mRNA function and localized translation. In the budding yeast, Saccharomyces cerevisiae, asymmetric mRNA transport localizes ~30 specific mRNAs including those encoding polarity and secretion factors, to the bud tip. The underlying process involves RNA-binding proteins (RBPs), molecular motors, processing bodies (PBs), and the actin cytoskeleton. Recently, pheromone a-factor expression in mating yeast was discovered to depend on proper localization of its mRNA, MFA2 mRNAs in conjunction with PBs cluster at the shmoo tip to form “mating bodies”, from which a-factor is locally expressed. The mechanism ensuring the correct targeting of mRNA to the shmoo tip is poorly understood. Here we analyzed the kinetics and trajectories of MFA2 mRNA transport in living, alpha-factor treated yeast. Two- (2D) and three-dimensional (3D) analyses allowed us to reconstruct the granule tracks and estimate granule velocities. Tracking analysis of single MFA2 mRNA granules, labeled using a fluorescent aptamer system, demonstrated three types movement: vibrational, oscillatory and translocational. The mRNA granule transport was complex; a granule could change its movement behavior and composition during its journey to the shmoo. Processing body assembly and the actin-based motor, Myo4p, were involved in movement of MFA2 mRNA to the shmoo, but neither was required, indicating that multiple mechanisms for translocation were at play. Our visualization studies present a dynamic view of the localization mechanism in shmoo-bearing cells.

The adaptor protein Ste50 directly modulates yeast MAPK signaling specificity through differential connections of its RA domain.

Discriminating among diverse environmental stimuli is critical for organisms to ensure their proper development, homeostasis, and survival. Saccharomyces cerevisiae regulates mating, osmoregulation, and filamentous growth using three different MAPK signaling pathways that share common components and therefore must ensure specificity. The adaptor protein Ste50 activates Ste11p, the MAP3K of all three modules. Its Ras association (RA) domain acts in both hyperosmolar and filamentous growth pathways, but its connection to the mating pathway is unknown. Genetically probing the domain, we found mutants that specifically disrupted mating or HOG-signaling pathways or both. Structurally these residues clustered on the RA domain, forming distinct surfaces with a propensity for protein–protein interactions. GFP fusions of wild-type (WT) and mutant Ste50p show that WT is localized to the shmoo structure and accumulates at the growing shmoo tip. The specifically pheromone response–defective mutants are severely impaired in shmoo formation and fail to localize ste50p, suggesting a failure of association and function of Ste50 mutants in the pheromone-signaling complex. Our results suggest that yeast cells can use differential protein interactions with the Ste50p RA domain to provide specificity of signaling during MAPK pathway activation.

Cell fusion in yeast is negatively regulated by components of the cell wall integrity pathway.

During mating, Saccharomyces cerevisiae cells must degrade the intervening cell wall to allow fusion of the partners. Because improper timing or location of cell wall degradation would cause lysis, the initiation of cell fusion must be highly regulated. Here, we find that yeast cell fusion is negatively regulated by components of the cell wall integrity (CWI) pathway. Loss of the cell wall sensor, MID2, specifically causes “mating-induced death” after pheromone exposure. Mating-induced death is suppressed by mutations in cell fusion genes (FUS1, FUS2, RVS161, CDC42), implying that mid2Δ cells die from premature fusion without a partner. Consistent with premature fusion, mid2Δ shmoos had thinner cell walls and lysed at the shmoo tip. Normally, Cdc42p colocalizes with Fus2p to form a focus only when mating cells are in contact (prezygotes) and colocalization is required for cell fusion. However, Cdc42p was aberrantly colocalized with Fus2p to form a focus in mid2Δ shmoos. A hyperactive allele of the CWI kinase Pkc1p (PKC1*) caused decreased cell fusion and Cdc42p localization in prezygotes. In shmoos, PKC1* increased Cdc42p localization; however, it was not colocalized with Fus2p or associated with cell death. We conclude that Mid2p and Pkc1p negatively regulate cell fusion via Cdc42p and Fus2p.

Kel1p Mediates Yeast Cell Fusion Through a Fus2p- and Cdc42p-Dependent Mechanism.

Cell fusion is ubiquitous among eukaryotes. Although little is known about the molecular mechanism, several proteins required for cell fusion in the yeast Saccharomyces cerevisiae have been identified. Fus2p, a key regulator of cell fusion, localizes to the shmoo tip in a highly regulated manner. C-terminal truncations of Fus2p cause mislocalization and fusion defects, which are suppressed by overexpression of Kel1p, a kelch-domain protein of unknown function previously implicated in cell fusion. We hypothesize that Fus2p mislocalization is caused by auto-inhibition, which is alleviated by Kel1p overexpression. Previous work showed that Fus2p localization is mediated by both Fus1p- and actin-dependent pathways. We show that the C-terminal mutations mainly affect the actin-dependent pathway. Suppression of the Fus2p localization defect by Kel1p is dependent upon Fus1p, showing that suppression does not bypass the normal pathway. Kel1p and a homolog, Kel2p, are required for efficient Fus2p localization, acting through the actin-dependent pathway. Although Kel1p overexpression can weakly suppress the mating defect of a FUS2 deletion, the magnitude of suppression is allele specific. Therefore, Kel1p augments, but does not bypass, Fus2p function. Fus2p mediates cell fusion by binding activated Cdc42p Although Kel1p overexpression suppresses a Cdc42p mutant that is defective for Fus2p binding, cell fusion remains dependent upon Fus2p These data suggest that Fus2p, Cdc42p, and Kel1p form a ternary complex, which is stabilized by Kel1p Supporting this hypothesis, Kel1p interacts with two domains of Fus2p, partially dependent on Cdc42p We conclude that Kel1p enhances the activity of Fus2p/Cdc42p in cell fusion.

An Amphiphysin-Like Domain in Fus2p Is Required for Rvs161p Interaction and Cortical Localization.

Cell–cell fusion fulfils essential roles in fertilization, development and tissue repair. In the budding yeast, Saccharomyces cerevisiae, fusion between two haploid cells of opposite mating type generates the diploid zygote. Fus2p is a pheromone-induced protein that regulates cell wall removal during mating. Fus2p shuttles from the nucleus to localize at the shmoo tip, bound to Rvs161p, an amphiphysin. However, Rvs161p independently binds a second amphiphysin, Rvs167p, playing an essential role in endocytosis. To understand the basis of the Fus2p–Rvs161p interaction, we analyzed Fus2p structural domains. A previously described N-terminal domain (NTD) is necessary and sufficient to regulate nuclear/cytoplasmic trafficking of Fus2p. The Dbl homology domain (DBH) binds GTP-bound Cdc42p; binding is required for cell fusion, but not localization. We identified an approximately 200 amino acid region of Fus2p that is both necessary and sufficient for Rvs161p binding. The Rvs161p binding domain (RBD) contains three predicted alpha-helices; structural modeling suggests that the RBD adopts an amphiphysin-like structure. The RBD contains a 13-amino-acid region, conserved with Rvs161p and other amphiphysins, which is essential for binding. Mutations in the RBD, predicted to affect membrane binding, abolish cell fusion without affecting Rvs161p binding. We propose that Fus2p/Rvs161p form a novel heterodimeric amphiphysin required for cell fusion. Rvs161p binding is required but not sufficient for Fus2p localization. Mutations in the C-terminal domain (CTD) of Fus2p block localization, but not Rvs161p binding, causing a significant defect in cell fusion. We conclude that the Fus2p CTD mediates an additional, Rvs161p-independent interaction at the shmoo tip.

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.

Flippase function is required for signaling competence in the mating pheromone response pathway

In the plasma membrane (PM) of eukaryotic cells, phospholipid asymmetry appears to be important for cell signaling and cytokinesis. The outer leaflet of the PM contains predominantly phosphatidylcholine and sphingolipids, whereas the inner leaflet is enriched in phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEth) and phosphatidylinositol and its phosphorylated derivative (PIP2). Members of the P‐type 4 ATPases (Flippase) maintain membrane asymmetry by translocating PtdSer and PtdEth from the outer leaflet to the cytosolic leaflet of the PM. In S. cerevisiae, five genes (DNF1, DNF2, DNF3, DRS2 and NEO1) encode flippases. In MATa cells responding to α‐factor, we found that Dnf1, Dnf2 and Dnf3, and a flippase activator (Fpk1 kinase), localize at the shmoo tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone MAPK cascade) is recruited. Moreover, a MATadnf1Δ dnf2Δ dnf3Δ triple mutant exhibits a reduction in its ability to respond to α‐factor due to a pronounced reduction in the stability of Ste5 resulting from an elevated rate of its Cln2·Cdc28 degradation. By contrast, a MATadnf1Δ dnf3Δ drs2Δ mutant also displays a reduction in its ability to respond to α‐factor, but in these cells the defect was attributable to inefficient recruitment of Ste5 to the PM because of severe mislocalization of the PtdIns4P and PIP2 pools.

Cdc42 Explores the Cell Periphery for Mate Selection in Fission Yeast

How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification.

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5- bis phosphate distribution is required for MAPK signaling

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P(2), we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P(2), stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P(2) is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P(2) necessary for the membrane anchoring and function of a signaling complex.

Structure of sterol aliphatic chains affects yeast cell shape and cell fusion during mating

Under mating conditions, yeast cells adopt a characteristic pear-shaped morphology, called a "shmoo," as they project a cell extension toward their mating partners. Mating partners make contact at their shmoo tips, dissolve the intervening cell wall, and fuse their plasma membranes. We identified mutations in ERG4, encoding the enzyme that catalyzes the last step of ergosterol biosynthesis, that impair both shmoo formation and cell fusion. Upon pheromone treatment, erg4Delta mutants polarized growth, lipids, and proteins involved in mating but did not form properly shaped shmoos and fused with low efficiency. Supplementation with ergosterol partially suppressed the shmooing defect but not the cell fusion defect. By contrast, removal of the Erg4 substrate ergosta-5,7,22,24(28)-tetraenol, which accumulates in erg4Delta mutant cells and contains an extra double bond in the aliphatic chain of the sterol, restored both shmooing and cell fusion to wild-type levels. Thus, a two-atom change in the aliphatic moiety of ergosterol is sufficient to obstruct cell shape remodeling and cell fusion.

Dynamic localization of yeast Fus2p to an expanding ring at the cell fusion junction during mating

Fus2p is a pheromone-induced protein associated with the amphiphysin homologue Rvs161p, which is required for cell fusion during mating in Saccharomyces cerevisiae. We constructed a functional Fus2p–green fluorescent protein (GFP), which exhibits highly dynamic localization patterns in pheromone-responding cells (shmoos): diffuse nuclear, mobile cytoplasmic dots and stable cortical patches concentrated at the shmoo tip. In mitotic cells, Fus2p-GFP is nuclear but becomes cytoplasmic as cells form shmoos, dependent on the Fus3p protein kinase and high levels of pheromone signaling. The rapid cytoplasmic movement of Fus2p-GFP dots requires Rvs161p and polymerized actin and is aberrant in mutants with compromised actin organization, which suggests that the Fus2p dots are transported along actin cables, possibly in association with vesicles. Maintenance of Fus2p-GFP patches at the shmoo tip cortex is jointly dependent on actin and a membrane protein, Fus1p, which suggests that Fus1p is an anchor for Fus2p. In zygotes, Fus2p-GFP forms a dilating ring at the cell junction, returning to the nucleus at the completion of cell fusion.

Afr1p has a role in regulating the localization of Mpk1p at the shmoo tip in Saccharomyces cerevisiae

Afr1p functions to promote adaptation to pheromone-induced growth arrest and morphogenesis. We show here that Afr1p regulates polarized localization of the Mpk1p MAP kinase in shmooing cells. Deletion of AFR1 results in mislocalization of Mpk1p although the scaffold protein Spa2p localizes normally at shmoo tip, and overexpression of Spa2 cannot rescue this defect, indicating Afr1p in required for Spa2p to recruit Mpk1 to the site of polarized growth during mating. Overexpression of SPA2 partially suppresses the morphogenetic defect of afr1Δ cells upon α-factor induction, suggesting the two proteins function in the same genetic pathway with Spa2p acts downstream of Afr1p.

Microtubule dynamics in the budding yeast mating pathway

In order for haploid gametes to fuse during fertilization, microtubules (MTs) must generate forces that are sufficient to move the nuclei together. Nuclear movements during fertilization rely on microtubule-associated proteins (MAPs), many of which have been characterized extensively during mitosis. A useful model system to study MT-dependent forces before nuclear fusion, or karyogamy, is the mating pathway of budding yeast. Dynamic MTs are guided to the mating projection (shmoo tip) when plus-end-binding proteins interact with polarized actin microfilaments. If two shmoo tips are in proximity they may fuse, dissolving the MT-cortical interactions. Subsequently, oppositely oriented MT plus ends interact and draw the nuclei together. The plus-end-binding proteins in the yeast mating pathway are conserved in metazoan cells and may play a role in higher eukaryotic fertilizaton. Thus, understanding the mechanism of plus end orientation and karyogamy in budding yeast will reveal mechanisms of MT-dependent force generation conserved throughout evolution.

Mdy2, a ubiquitin-like (UBL)-domain protein, is required for efficient mating in Saccharomyces cerevisiae

MDY2, a gene required for efficient mating of the yeast Saccharomyces cerevisiae, was characterized in this study. The gene encodes a protein of 212 amino acids, which contains a ubiquitin-like (UBL) domain (residues 74-149). Deletion of MDY2 is associated with a five- to sevenfold reduction in mating efficiency, mainly due to defects in nuclear migration and karyogamy at the prezygotic stage. However, prior to mating pair fusion, shmoo formation is reduced by 30%, with a concomitant failure to form mating pairs. Strikingly, migration of the nucleus into the shmoo tip is also delayed or fails to occur. In addition, we show that in mdy2 mutants, microtubule bundles, as well as the microtubule end-binding protein Kar9, fail to localize properly to the shmoo tip, suggesting that the nuclear migration defect could be due to aberrant localization of Kar9. Pheromone signal transduction (as measured by FUS1 induction by alpha-factor) is not affected in mdy2delta mutants and mitosis is also normal in these cells. MDY2 is not induced by mating pheromone. In vegetatively growing cells, GFP-Mdy2 is localized in the nucleus, and remains nuclear after exposure of cells to alpha-factor. His-tagged Mdy2 shows no evidence of the C-terminal processing typical of ubiquitin, and also localizes to the nucleus. Thus MDY2 is a novel gene, whose product plays a role in shmoo formation and in nuclear migration in the pre-zygote, possibly by interacting with other UBL-type proteins that possess ubiquitin association (UBA) domains.

Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end–binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo.

The Bud14p-Glc7p complex functions as a cortical regulator of dynein in budding yeast.

Regulated interactions between microtubules (MTs) and the cell cortex control MT dynamics and position the mitotic spindle. In eukaryotic cells, the adenomatous polyposis coli/Kar9p and dynein/dynactin pathways are involved in guiding MT plus ends and MT sliding along the cortex, respectively. Here we identify Bud14p as a novel cortical activator of the dynein/dynactin complex in budding yeast. Bud14p accumulates at sites of polarized growth and the mother-bud neck during cytokinesis. The localization to bud and shmoo tips requires an intact actin cytoskeleton and the kelch-domain-containing proteins Kel1p and Kel2p. While cells lacking Bud14p function fail to stabilize the pre-anaphase spindle at the mother-bud neck, overexpression of Bud14p is toxic and leads to elongated astral MTs and increased dynein-dependent sliding along the cell cortex. Bud14p physically interacts with the type-I phosphatase Glc7p, and localizes Glc7p to the bud cortex. Importantly, the formation of Bud14p-Glc7p complexes is necessary to regulate MT dynamics at the cortex. Taken together, our results suggest that Bud14p functions as a regulatory subunit of the Glc7p type-I phosphatase to stabilize MT interactions specifically at sites of polarized growth.

Cik1 Targets the Minus-End Kinesin Depolymerase Kar3 to Microtubule Plus Ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.

Lipid raft polarization contributes to hyphal growth in Candida albicans.

The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeast Saccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA 99:14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogen Candida albicans to grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol polarization and hyphal morphogenesis in that both were disrupted by low concentrations of latrunculin A that did not prevent budding. Furthermore, blocking either sphingolipid biosynthesis with myriocin or sterol biosynthesis with ketoconazole resulted in a loss of ergosterol polarization and caused abnormal hyphal morphogenesis, suggesting that lipid rafts are involved. Since hyphal growth is required for the full virulence of C. albicans, these results suggest that membrane polarization may contribute to the pathogenesis of this organism.

Slow Diffusion of Proteins in the Yeast Plasma Membrane Allows Polarity to Be Maintained by Endocytic Cycling

Many cells show a polarized distribution of some plasma membrane proteins, which may be maintained either by a diffusion barrier [1] or kinetically: as first demonstrated in fibroblasts [2], locally exocytosed proteins will remain polarized if they are endocytosed and recycled before they can diffuse to equilibrium. In yeast, actin cables direct exocytosis to the bud and to the tips of polarized mating intermediates termed shmoos [3, 4]. A septin ring at the bud neck retains some proteins [5, 6], but shmoos lack this. Here, we show that the exocytic SNARE Snc1 is kinetically polarized. It is concentrated at bud and shmoo tips, and this requires its endocytosis. Kinetic polarization is possible in these small cells because proteins diffuse much more slowly in the yeast plasma membrane than would be expected from measurements in animal cells. Slow diffusion requires neither the cell wall nor polymerized actin, but it is affected in the ergosterol synthesis mutant erg6. Other proteins also require endocytosis for efficient polarization, and the plasma membrane SNARE Sso1 can be polarized merely by appending an endocytic signal. Thus, despite their small size, yeast cells can use localized exocytosis and endocytic recycling as a simple mechanism to maintain polarity.