Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain.

Clathrin facilitates vesicle formation during endocytosis and sorting in the trans-Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc-YR) , which fused the N-terminus of yeast CHC (1-1312) to the rat CHC residues 1318-1675, including the CHC trimerization region. The novel CHC-YR allele encoded a stable protein that fractionated as a trimer. CHC-YR also complemented chc1Δ slow growth and clathrin TGN/endosomal sorting defects. In strains depleted for Clc1 (either clc1Δ or chc1Δ clc1Δ), CHC-YR, but not CHC1, suppressed TGN/endosomal sorting and growth phenotypes. Chc-YR-GFP (green fluorescent protein) localized to the TGN and cortical patches on the plasma membrane, like Chc1 and Clc1. However, Clc1-GFP was primarily cytoplasmic in chc1Δ cells harboring pCHC-YR, indicating that Chc-YR does not bind yeast CLC. Still, some partial phenotypes persisted in cells with Chc-YR, which are likely due either to loss of CLC recruitment or chimeric HC lattice instability. Ultimately, these studies have created a tool to examine non-trimerization roles for the clathrin LC.

Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization.

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.

Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes.

Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking.

Getting in touch with the clathrin terminal domain.

The N-terminal domain (TD) of the clathrin heavy chain is folded into a seven-bladed ß-propeller that projects inward from the polyhedral outer clathrin coat. As the most membrane-proximal portion of assembled clathrin, the TD is a major protein-protein interaction node. Contact with the TD ß-propeller occurs through short peptide sequences typically located within intrinsically disordered segments of coat components that usually are elements of the membrane-apposed, inner 'adaptor' coat layer. A huge variation in TD-binding motifs is known and now four spatially discrete interaction surfaces upon the ß-propeller have been delineated. An important operational feature of the TD interaction sites in vivo is functional redundancy. The recent discovery that 'pitstop' chemical inhibitors apparently occupy only one of the four TD interaction surfaces, but potently block clathrin-mediated endocytosis, warrants careful consideration of the underlying molecular basis for this inhibition.

Novel interaction between the co-chaperone Cdc37 and Rho GTPase exchange factor Vav3 promotes androgen receptor activity and prostate cancer growth.

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR. We sought to identify Vav3 binding partners in an effort to understand the molecular mechanisms underlying Vav3 enhancement of AR activity and to identify new therapeutic targets. The cell division cycle 37 homolog (Cdc37), a protein kinase-specific co-chaperone for Hsp90, was identified as a Vav3 interacting protein by yeast two-hybrid screening. Vav3-Cdc37 interaction was confirmed by GST pulldown and, for native proteins, by co-immunoprecipitation experiments in prostate cancer cells. Cdc37 potentiated Vav3 co-activation of AR transcriptional activity and Vav3 enhancement of AR N-terminal-C-terminal interaction, which is essential for optimal receptor transcriptional activity. Cdc37 increased prostate cancer cell proliferation selectively in Vav3-expressing cells. Cdc37 did not affect Vav3 nucleotide exchange activity, Vav3 protein levels, or subcellular localization. Disruption of Vav3-Cdc37 interaction inhibited Vav3 enhancement of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer.

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.

Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis.

Phosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1 and Ark1 phosphorylate factors after vesicle internalization leading to coat disassembly. Scd5, a protein phosphatase-1 (PP1)-targeting subunit, is proposed to regulate dephosphorylation of Prk1/Ark1 substrates to promote new rounds of endocytosis. In this study we analyzed scd5-PP1Δ2, a mutation causing impaired PP1 binding. scd5-PP1Δ2 caused hyperphosphorylation of several Prk1 endocytic targets. Live-cell imaging of 15 endocytic components in scd5-PP1Δ2 revealed that most factors arriving before the invagination/actin phase of endocytosis had delayed lifetimes. Severely affected were early factors and Sla2 (Hip1R homolog), whose lifetime was extended nearly fourfold. In contrast, the lifetime of Sla1, a Prk1 target, was extended less than twofold, but its cortical recruitment was significantly reduced. Delayed Sla2 dynamics caused by scd5-PP1Δ2 were suppressed by SLA1 overexpression. This was dependent on the LxxQxTG repeats (SR) of Sla1, which are phosphorylated by Prk1 and bind Pan1, another Prk1 target, in the dephosphorylated state. Without the SR, Sla1ΔSR was still recruited to the cell surface, but was less concentrated in cortical patches than Pan1. sla1ΔSR severely impaired endocytic progression, but this was partially suppressed by overexpression of LAS17, suggesting that without the SR region the SH3 region of Sla1 causes constitutive negative regulation of Las17 (WASp). These results demonstrate that Scd5/PP1 is important for recycling Prk1 targets to initiate new rounds of endocytosis and provide new mechanistic information on the role of the Sla1 SR domain in regulating progression to the invagination/actin phase of endocytosis.

Atg27p co-fractionates with clathrin-coated vesicles in budding yeast.

Atg27p, a single-pass transmembrane protein that functions in autophagy, localizes to a variety of cellular compartments including the pre-autophagosomal structure, late Golgi, vacuolar membrane, as well as early and late endosomes. Its cytoplasmic C-terminus contains a tyrosine sorting motif that allows for its transport to the vacuolar membrane and an additional sequence that allows for its retrieval from the vacuolar membrane to the endosome. Since clathrin is well known to mediate vesicular transport in the endomembrane system, the trafficking of Atg27p and its tyrosine sorting motif suggested that it might be trafficked inside clathrin-coated vesicles (CCVs). In our previous studies, Atg27p was identified by mass spectrometry as a potential component in CCVs, as it was present in CCVs isolated from both WT and auxilin-depleted cells. We now confirm that Atg27p is a component of CCVs using immunoblotting and additional mass spectrometry data.

Atg27p localization is clathrin- and Ent3p/5p-dependent.

The autophagy-related protein Atg27p has been previously shown to localize to the autophagy-specific pre-autophagosomal structure (PAS) as well as to several organelles, including the late Golgi, the vacuolar membrane, and the endosome. Given that Atg27p localization to the vacuolar membrane in particular has been shown to be dependent on both its C-terminal tyrosine sorting motif and the AP-3 adaptor, and that Atg27p can be found in clathrin-coated vesicles, we set out to determine whether Atg27p localization inside cells is dependent on clathrin or on any of its cargo adaptors. We report that Atg27p localization is clathrin- and Ent3p/5p-dependent.

In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast.

Clathrin-mediated transport is a major pathway for endocytosis. However, in yeast, where cortical actin patches are essential for endocytosis, plasma membrane-associated clathrin has never been observed. Using live cell imaging, we demonstrate cortical clathrin in association with the actin-based endocytic machinery in yeast. Fluorescently tagged clathrin is found in highly mobile internal trans-Golgi/endosomal structures and in smaller cortical patches. Total internal reflection fluorescence microscopy showed that cortical patches are likely endocytic sites, as clathrin is recruited prior to a burst of intensity of the actin patch/endocytic marker, Abp1. Clathrin also accumulates at the cortex with internalizing alpha factor receptor, Ste2p. Cortical clathrin localizes with epsins Ent1/2p and AP180s, and its recruitment to the surface is dependent upon these adaptors. In contrast, Sla2p, End3p, Pan1p, and a dynamic actin cytoskeleton are not required for clathrin assembly or exchange but are required for the mobility, maturation, and/or turnover of clathrin-containing endocytic structures.

L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1.

Dynamic regulation of the cell surface expression of adhesion molecules is an important mechanism for controlling neuronal growth cone motility and guidance. Clathrin-mediated vesicular internalization of L1 via the tyrosine-based endocytosis motif YRSL regulates adhesion and signaling by this Ig superfamily molecule. Here, we present evidence that tyrosine-1176 (Y1176) of the YRSL motif is phosphorylated in vivo. The nonreceptor tyrosine kinase (p60src) is implicated in L1-mediated neurite outgrowth, and we find that p60src phosphorylates Y1176 in vitro. Phosphorylation of Y1176 prevents L1 binding to AP-2, an adaptor required for clathrin-mediated internalization of L1. mAb 74-5H7 recognizes the sequence immediately NH2-terminal to the tyrosine-based motif and binds L1 only when Y1176 is dephosphorylated. 74-5H7 identifies a subset of L1 present at points of cell-cell contact and in vesicle-like structures that colocalize with an endocytosis marker. L1-L1 binding or L1 cross-linking induces a rapid increase in 74-5H7 immunoreactivity. Our data suggest a model in which homophilic binding or L1 cross-linking triggers transient dephosphorylation of the YRSL motif that makes L1 available for endocytosis. Thus, the regulation of L1 endocytosis through dephosphorylation of Y1176 is a critical regulatory point of L1-mediated adhesion and signaling.

Cortical recruitment and nuclear-cytoplasmic shuttling of Scd5p, a protein phosphatase-1-targeting protein involved in actin organization and endocytosis.

Scd5p regulates endocytosis and cortical actin organization as a targeting subunit for the Ser/Thr protein phosphatase-1 (PP1) in yeast. To identify localization signals in Scd5p required for cell surface recruitment, visualization of GFP-tagged Scd5 truncations and deletions was performed. Scd5p contains a PP1 binding site, a 3-repeat region of 20 amino acids (3R), and a 9-repeat region of 12 amino acids (9R). We found that the 9R is critical for cortical localization of Scd5p, but cortical recruitment is not essential for Scd5p's function in actin organization and endocytosis. We propose that Scd5p can target PP1 to endocytic factors in the cytoplasm that have been disassembled and/or inactivated by phosphorylation. We also found that Scd5p undergoes nuclear-cytoplasmic shuttling in a Crm1p-dependent manner. Scd5p-DeltaCT lacking the 9R region and its nuclear export signal (NES) accumulates in the nucleus, causing cortical actin and endocytic defects. Cytoplasmic localization and function of Scd5p-DeltaCT is restored by NES addition. However, removal of Scd5p's nuclear localization signal prevents nuclear entry, but endocytosis and actin organization remain relatively normal. These results indicate that nuclear-cytoplasmic shuttling is not required for regulation of Scd5p's cortical function and suggest that Scd5p has an independent nuclear function.

Novel function of clathrin light chain in promoting endocytic vesicle formation.

Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.

Identification of Suppressor of Clathrin Deficiency-1 (SCD1) and Its Connection to Clathrin-Mediated Endocytosis in Saccharomyces cerevisiae.

Clathrin is a major coat protein involved in vesicle formation during endocytosis and transport in the endosomal/trans Golgi system. Clathrin is required for normal growth of yeast (Saccharomyces cerevisiae) and in some genetic backgrounds deletion of the clathrin heavy chain gene (CHC1) is lethal. Our lab defined a locus referred to as " s uppressor of c lathrin d eficiency" (SCD1). In the presence of the scd1-v allele ("v" - viable), yeast cells lacking clathrin heavy chain survive but grow slowly, are morphologically abnormal and have many membrane trafficking defects. In the presence of scd1-i ("i"- inviable), chc1∆ causes lethality. As a strategy to identify SCD1, we used pooled linkage analysis and whole genome sequencing. Here, we report that PAL2 (YHR097C) is the SCD1 locus. pal2∆ is synthetic lethal with chc1∆; whereas a deletion of its paralog, PAL1, is not synthetic lethal with clathrin deficiency. Like Pal1, Pal2 has two NPF motifs that are potential binding sites for EH domain proteins such as the early endocytic factor Ede1, and Pal2 associates with Ede1 Also, GFP-tagged Pal2p localizes to cortical patches containing other immobile phase endocytic coat factors. Overall, our data show that PAL2 is the SCD1 locus and the Pal2 protein has characteristics of an early factor involved in clathrin-mediated endocytosis.

The actin-regulating kinase Prk1p negatively regulates Scd5p, a suppressor of clathrin deficiency, in actin organization and endocytosis.

Endocytosis is a dynamic process requiring a network of interacting proteins that assemble and disassemble during cargo capture and vesicle formation. A major mechanism for regulation of this process involves the reversible phosphorylation of endocytic factors. Recently, members of a new kinase family, the Ark/Prk kinases, which include mammalian AAK1 and GAK as well as yeast Prk1p, Ark1p, and Akl1p, were shown to regulate components of the endocytic machinery. These include animal AP-1/AP-2 mu chains and yeast Pan1p (Eps15-like), Sla1p, and epsins, but other potential targets are likely. SCD5, an essential yeast gene, was identified as a suppressor of clathrin deficiency. We also showed that Scd5p is required for normal cortical actin organization and endocytosis, possibly as a targeting subunit for protein phosphatase type 1 (PP1). Scd5p contains a central triple repeat (3R) motif related to a known Prk1p consensus phosphorylation site L/IxxQxTG, except that Q is replaced by T. In this study we demonstrate that the Scd5p 3R sequence is phosphorylated by Prk1p to negatively regulate Scd5p. Furthermore, we show that Prk1p, Ark1p, and Akl1p have different substrate specificities and play distinct roles in actin organization and endocytosis.

Scd5p and clathrin function are important for cortical actin organization, endocytosis, and localization of sla2p in yeast.

SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but an scd5-Delta338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Delta338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Delta338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars. scd5-Delta338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Delta338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells.

Auxilin facilitates membrane traffic in the early secretory pathway.

Coat protein complexes contain an inner shell that sorts cargo and an outer shell that helps deform the membrane to give the vesicle its shape. There are three major types of coated vesicles in the cell: COPII, COPI, and clathrin. The COPII coat complex facilitates vesicle budding from the endoplasmic reticulum (ER), while the COPI coat complex performs an analogous function in the Golgi. Clathrin-coated vesicles mediate traffic from the cell surface and between the trans-Golgi and endosome. While the assembly and structure of these coat complexes has been extensively studied, the disassembly of COPII and COPI coats from membranes is less well understood. We describe a proteomic and genetic approach that connects the J-domain chaperone auxilin, which uncoats clathrin-coated vesicles, to COPII and COPI coat complexes. Consistent with a functional role for auxilin in the early secretory pathway, auxilin binds to COPII and COPI coat subunits. Furthermore, ER-Golgi and intra-Golgi traffic is delayed at 15°C in swa2Δ mutant cells, which lack auxilin. In the case of COPII vesicles, we link this delay to a defect in vesicle fusion. We propose that auxilin acts as a chaperone and/or uncoating factor for transport vesicles that act in the early secretory pathway.

Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast.

14-3-3 proteins bind to phosphorylated proteins and regulate a variety of cellular activities as effectors of serine/threonine phosphorylation. To define processes requiring 14-3-3 function in yeast, mutants with increased sensitivity to reduced 14-3-3 protein levels were identified by synthetic lethal screening. One mutation was found to be allelic to YPK1, which encodes a Ser/Thr protein kinase. Loss of Ypk function causes hypersensitivity to rapamycin, similar to 14-3-3 mutations and other mutations affecting the TOR signaling pathway in yeast. Similar to treatment with rapamycin, loss of Ypk function disrupted translation, at least in part by causing depletion of eIF4G, a central adaptor protein required for cap-dependent mRNA translation initiation. In addition, Ypk1 as well as eIF4G protein levels were rapidly depleted upon nitrogen starvation, but not during glucose starvation, even though both conditions inhibit translation initiation. These results suggest that Ypk regulates translation initiation in response to nutrient signals, either through the TOR pathway or in a functionally related pathway parallel to TOR.

Lessons from yeast for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. Half a century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process. As CME is highly conserved from yeast to humans, budding yeast provides an evolutionary template for this process and has been a valuable system for dissecting the underlying molecular mechanisms. In this review we trace the formation of a clathrin-coated vesicle from initiation to uncoating, focusing on key findings from the yeast system.