Spindle Position Checkpoint Kinase Kin4 Regulates Organelle Transport in Saccharomyces cerevisiae.

Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes. Studies in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) revealed that a block in actomyosin-dependent transport affects organelle inheritance to daughter cells. Indeed, class V Myosins, Myo2, and Myo4, and many of their organelle receptors, have been identified as key factors in organelle inheritance. However, the spatiotemporal regulation of yeast organelle transport remains poorly understood. Using peroxisome inheritance as a proxy to study actomyosin-based organelle transport, we performed an automated genome-wide genetic screen in S. cerevisiae. We report that the spindle position checkpoint (SPOC) kinase Kin4 and, to a lesser extent, its paralog Frk1, regulates peroxisome transport, independent of their role in the SPOC. We show that Kin4 requires its kinase activity to function and that both Kin4 and Frk1 protect Inp2, the peroxisomal Myo2 receptor, from degradation in mother cells. In addition, vacuole inheritance is also affected in kin4/frk1-deficient cells, suggesting a common regulatory mechanism for actin-based transport for these two organelles in yeast. More broadly our findings have implications for understanding actomyosin-based transport in cells.

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2 )2 (tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

A Ruthenium(II) Polypyridyl Complex Disrupts Actin Cytoskeleton Assembly and Blocks Cytokinesis.

The dinuclear RuII complex [(Ru(phen)2)2(tpphz)]4+ (phen=1,10-phenanthroline, tpphz=tetrapyridophenazine) "RuRuPhen" blocks the transformation of G-actin monomers to F-actin filaments with no disassembly of pre-formed F-actin. Molecular docking studies indicate multiple RuRuPhen molecules bind to the surface of G-actin but not the binding pockets of established actin polymerisation inhibitors. In cells, addition of RuRuPhen causes rapid disruption to actin stress fibre organisation, compromising actomyosin contractility and cell motility; due to this effect RuRuPhen interferes with late-stage cytokinesis. Immunofluorescent microscopy reveals that RuRuPhen causes cytokinetic abscission failure by interfering with endosomal sorting complexes required for transport (ESCRT) complex recruitment.

The Pex3-Inp1 complex tethers yeast peroxisomes to the plasma membrane.

A subset of peroxisomes is retained at the mother cell cortex by the Pex3-Inp1 complex. We identify Inp1 as the first known plasma membrane-peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria that a contact site tether protein must adhere to. We show that Inp1 is present in the correct subcellular location to interact with both the plasma membrane and peroxisomal membrane and has the structural and functional capacity to be a PM-PER tether. Additionally, expression of artificial PM-PER tethers is sufficient to restore retention in inp1Δ cells. We show that Inp1 mediates peroxisome retention via an N-terminal domain that binds PI(4,5)P2 and a C-terminal Pex3-binding domain, forming a bridge between the peroxisomal membrane and the plasma membrane. We provide the first molecular characterization of the PM-PER tether and show it anchors peroxisomes at the mother cell cortex, suggesting a new model for peroxisome retention.

Mutation of key lysine residues in the Insert B region of the yeast dynamin Vps1 disrupts lipid binding and causes defects in endocytosis.

The yeast dynamin-like protein Vps1 has roles at multiple stages of membrane trafficking including Golgi to vacuole transport, endosomal recycling, endocytosis and in peroxisomal fission. While the majority of the Vps1 amino acid sequence shows a high level of identity with the classical mammalian dynamins, it does not contain a pleckstrin homology domain (PH domain). The Dyn1 PH domain has been shown to bind to lipids with a preference for PI(4,5)P2 and it is considered central to the function of Dyn1 in endocytosis. The lack of a PH domain in Vps1 has raised questions as to whether the protein can function directly in membrane fusion or fission events. Here we demonstrate that the region Insert B, located in a position equivalent to the dynamin PH domain, is able to bind directly to lipids and that mutation of three lysine residues reduces its capacity to interact with lipids, and in particular with PI(4,5)P2. The Vps1 KKK-AAA mutant shows more diffuse staining but does still show some localization to compartments adjacent to vacuoles and to endocytic sites suggesting that other factors are also involved in its recruitment. This mutant selectively blocks endocytosis, but is functional in other processes tested. While mutant Vps1 can localise to endocytic sites, the mutation results in a significant increase in the lifetime of the endocytic reporter Sla2 and a high proportion of defective scission events. Together our data indicate that the lipid binding capacity of the Insert B region of Vps1 contributes to the ability of the protein to associate with membranes and that its capacity to interact with PI(4,5)P2 is important in facilitating endocytic scission.

Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.

Disruption of the plant-specific CFS1 gene impairs autophagosome turnover and triggers EDS1-dependent cell death.

Cell death, autophagy and endosomal sorting contribute to many physiological, developmental and immunological processes in plants. They are mechanistically interconnected and interdependent, but the molecular basis of their mutual regulation has only begun to emerge in plants. Here, we describe the identification and molecular characterization of CELL DEATH RELATED ENDOSOMAL FYVE/SYLF PROTEIN 1 (CFS1). The CFS1 protein interacts with the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT I (ESCRT-I) component ELCH (ELC) and is localized at ESCRT-I-positive late endosomes likely through its PI3P and actin binding SH3YL1 Ysc84/Lsb4p Lsb3p plant FYVE (SYLF) domain. Mutant alleles of cfs1 exhibit auto-immune phenotypes including spontaneous lesions that show characteristics of hypersensitive response (HR). Autoimmunity in cfs1 is dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-mediated effector-triggered immunity (ETI) but independent from salicylic acid. Additionally, cfs1 mutants accumulate the autophagy markers ATG8 and NBR1 independently from EDS1. We hypothesize that CFS1 acts at the intersection of autophagosomes and endosomes and contributes to cellular homeostasis by mediating autophagosome turnover.

Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.

WASP family proteins, more than Arp2/3 activators.

Wiskott-Aldrich syndrome protein (WASP) family proteins have been extensively characterized as factors that promote the nucleation of actin through the activation of the protein complex Arp2/3. While yeast mostly have a single member of the family, mammalian cells have at least six different members, often with multiple isoforms. Members of the family are characterized by a common structure. Their N-termini are varied and are considered to confer spatial and temporal regulation of Arp2/3-activating activity, whereas their C-terminal half contains a polyproline-rich region, one or more WASP homology-2 (WH2) actin-binding domains and motifs that bind directly to Arp2/3. Recent studies, however, indicate that the yeast WASP homologue Las17 is able to nucleate actin independently of Arp2/3 through the function of novel G-actin-binding activities in its polyproline region. This allows Las17 to generate the mother filaments that are needed for subsequent Arp2/3 recruitment and activation during the actin polymerization that drives endocytic invagination in yeast. In this review, we consider how motifs within the polyproline region of Las17 support nucleation of actin filaments, and whether similar mechanisms might exist among other family members.

Elucidating Key Motifs Required for Arp2/3-Dependent and Independent Actin Nucleation by Las17/WASP.

Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. Arp2/3 is a well-characterised protein complex that can promote nucleation of new filaments, though its activity requires additional nucleation promotion factors (NPFs). The best recognized of these factors are the WASP family of proteins that contain binding motifs for both monomeric actin and for Arp2/3. Previously we demonstrated that the yeast WASP homologue, Las17, in addition to activating Arp2/3 can also nucleate actin filaments de novo, independently of Arp2/3. This activity is dependent on its polyproline rich region. Through biochemical and in vivo analysis we have now identified key motifs within the polyproline region that are required for nucleation and elongation of actin filaments, and have addressed the role of the WH2 domain in the context of actin nucleation without Arp2/3. We have also demonstrated that full length Las17 is able to bind liposomes giving rise to the possibility of direct linkage of nascent actin filaments to specific membrane sites to which Las17 has been recruited. Overall, we propose that Las17 functions as the key initiator of de novo actin filament formation at endocytic sites by nucleating, elongating and tethering nascent filaments which then serve as a platform for Arp2/3 recruitment and function.

Insights into dynamin-associated disorders through analysis of equivalent mutations in the yeast dynamin Vps1.

The dynamins represent a superfamily of proteins that have been shown to function in a wide range of membrane fusion and fission events. An increasing number of mutations in the human classical dynamins, Dyn-1 and Dyn-2 has been reported, with diseases caused by these changes ranging from Charcot-Marie-Tooth disorder to epileptic encephalopathies. The budding yeast, Saccharomyces cerevisiae expresses a single dynamin-related protein that functions in membrane trafficking, and is considered to play a similar role to Dyn-1 and Dyn-2 during scission of endocytic vesicles at the plasma membrane. Large parts of the dynamin protein are highly conserved across species and this has enabled us in this study to select a number of disease causing mutations and to generate equivalent mutations in Vps1. We have then studied these mutants using both cellular and biochemical assays to ascertain functions of the protein that have been affected by the changes. Specifically, we demonstrate that the Vps1-G397R mutation (Dyn-2 G358R) disrupts protein oligomerization, Vps1-A447T (Dyn-1 A408T) affects the scission stage of endocytosis, while Vps1-R298L (Dyn-1 R256L) affects lipid binding specificity and possibly an early stage in endocytosis. Overall, we consider that the yeast model will potentially provide an avenue for rapid analysis of new dynamin mutations in order to understand the underlying mechanisms that they disrupt.

Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1.

The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process.

A Charge Swap mutation E461K in the yeast dynamin Vps1 reduces endocytic invagination.

Vps1 is the yeast dynamin-like protein that functions during several membrane trafficking events including traffic from Golgi to vacuole, endosomal recycling and endocytosis. Vps1 can also function in peroxisomal fission indicating that its ability to drive membrane fission is relatively promiscuous. It has been of interest therefore that several mutations have been identified in Vps1 that only disrupt its endocytic function. Most recently, disruption of the interaction with actin through mutation of residues in one of the central stalk α helices (RR457,458 EE) has been shown to disrupt endocytosis and cause an accumulation of highly elongated invaginations in cells. This data supports the idea that an interaction between Vps1 and actin is important to drive the scission stage in endocytosis. Another Vps1 mutant generated in the study was vps1 E461K. Here we show data demonstrating that the E461K mutation also disrupts endocytosis but at an early stage, resulting in inhibition of the invagination step itself.

Distinct Actin and Lipid Binding Sites in Ysc84 Are Required during Early Stages of Yeast Endocytosis.

During endocytosis in S. cerevisiae, actin polymerization is proposed to provide the driving force for invagination against the effects of turgor pressure. In previous studies, Ysc84 was demonstrated to bind actin through a conserved N-terminal domain. However, full length Ysc84 could only bind actin when its C-terminal SH3 domain also bound to the yeast WASP homologue Las17. Live cell-imaging has revealed that Ysc84 localizes to endocytic sites after Las17/WASP but before other known actin binding proteins, suggesting it is likely to function at an early stage of membrane invagination. While there are homologues of Ysc84 in other organisms, including its human homologue SH3yl-1, little is known of its mode of interaction with actin or how this interaction affects actin filament dynamics. Here we identify key residues involved both in Ysc84 actin and lipid binding, and demonstrate that its actin binding activity is negatively regulated by PI(4,5)P2. Ysc84 mutants defective in their lipid or actin-binding interaction were characterized in vivo. The abilities of Ysc84 to bind Las17 through its C-terminal SH3 domain, or to actin and lipid through the N-terminal domain were all shown to be essential in order to rescue temperature sensitive growth in a strain requiring YSC84 expression. Live cell imaging in strains with fluorescently tagged endocytic reporter proteins revealed distinct phenotypes for the mutants indicating the importance of these interactions for regulating key stages of endocytosis.

A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

Function and interactions of the Ysc84/SH3yl1 family of actin- and lipid-binding proteins.

Understanding how actin filaments are nucleated, polymerized and disassembled in close proximity to cell membranes is an area of growing interest. Protrusion of the plasma membrane is required for cell motility, whereas inward curvature or invagination is required for endocytic events. These morphological changes in membrane are often associated with rearrangements of actin, but how the many actin-binding proteins of eukaryotes function in a co-ordinated way to generate the required responses is still not well understood. Identification and analysis of proteins that function at the interface between the plasma membrane and actin-regulatory networks is central to increasing our knowledge of the mechanisms required to transduce the force of actin polymerization to changes in membrane morphology. The Ysc84/SH3yl1 proteins have not been extensively studied, but work in both yeast and mammalian cells indicate that these proteins function at the hub of networks integrating regulation of filamentous actin (F-actin) with changes in membrane morphology.

Apm4, the mu subunit of yeast AP-2 interacts with Pkc1, and mutation of the Pkc1 consensus phosphorylation site Thr176 inhibits AP-2 recruitment to endocytic sites.

The AP-2 endocytic adaptor has been extensively characterized in mammalian cells and is considered to play a role both in cargo binding and in formation of endocytic sites. However, despite our detailed knowledge of mechanistic aspects of endocytic complex assembly and disassembly in the model organism Saccharomyces cerevisiae, no function of AP-2 had been described in wild-type yeast under normal growth conditions. A recent study however revealed that disruption of the complex caused by deletion of the gene encoding its mu subunit (APM4) caused defects in cell polarity such that responses to pheromone, nutritional status and cell wall damage were affected. Furthermore, a homozygous deletion of the mu subunit gene in Candida albicans affected its ability to grow hyphae. Direct binding to the yeast cell wall stress sensor Mid2 was detected, and in an apm4 deletion strain Mid2 showed reduced re-localization to the mother bud neck region following cell wall damage with calcofluor or to the mating projection tip. Here we demonstrate an interaction between Apm4 and the yeast cell wall integrity pathway component Pkc1 and show that mutation of the predicted Pkc1 site in the Apm4 hinge region affects recruitment of the AP-2 complex to endocytic sites.

An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.

Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarized cell responses.

The AP-2 complex is a heterotetrameric endocytic cargo-binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin-mediated endocytosis. While budding yeast has clear homologues of all four AP-2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP-2 has remained enigmatic. Here, we demonstrate that AP-2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae. Deletion of APM4, the cargo-binding mu subunit of AP-2, causes defects in pseudohyphal growth, generation of a mating projection and the cell wall damage response. In an apm4 null mutant, the cell wall stress sensor Mid2 is unable to relocalize to the tip of a mating projection following pheromone addition, or to the mother bud neck in response to cell wall damage. A direct binding interaction between Mid2 and the mu homology domain of Apm4 further supports a model in which AP-2 binds Mid2 to facilitate its internalization and relocalization in response to specific signals. Thus, Mid2 is the first cargo for AP-2 identified in yeast. We propose that endocytic recycling of Mid2 and other components is required for polarized cell responses ensuring cell wall deposition and is tightly monitored during cell growth.