Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae

The internalization step of endocytosis in yeast requires actin and sterols for maximum efficiency. In addition, many receptors and plasma membrane proteins must be phosphorylated and ubiquitylated prior to internalization. The Saccharomyces cerevisiae end8-1 mutant is allelic to lcb1, a mutant defective in the first step of sphingoid base synthesis. Upon arrest of sphingoid base synthesis a rapid block in endocytosis is seen. This block can be overcome by exogenous sphingoid base. Under conditions where endogenous sphingosine base synthesis was blocked and exogenous sphingoid bases could not be converted to phosphorylated sphingoid bases or to ceramide, sphingoid bases could still suppress the endocytic defect. Therefore, the required lipid is most likely a sphingoid base. Interestingly, sphingoid base synthesis is required for proper actin organization, but is not required for receptor phosphorylation. This is the first case of a physiological role for sphingoid base synthesis, other than as a precursor for ceramide or phosphorylated sphingoid base synthesis.     

The conserved Pkh-Ypk kinase cascade is required for endocytosis in yeast.

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast alpha-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base-regulated serine-threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum- and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in alpha-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in alpha-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.     

The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.     

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).     

NPFXD-mediated endocytosis is required for polarity and function of a yeast cell wall stress sensor

The actin-associated protein Sla1p, through its SHD1 domain, acts as an adaptor for the NPFX(1,2)D endocytic targeting signal in yeast. Here we report that Wsc1p, a cell wall stress sensor, depends on this signal-adaptor pair for endocytosis. Mutation of NPFDD in Wsc1p or expression of Sla1p lacking SHD1 blocked Wsc1p internalization. By live cell imaging, endocytically defective Wsc1p was not concentrated at sites of endocytosis. Polarized distribution of Wsc1p to regions of cell growth was lost in the absence of endocytosis. Mutations in genes necessary for endosome to Golgi traffic caused redistribution of Wsc1p from the cell surface to internal compartments, indicative of recycling. Inhibition of Wsc1p endocytosis caused defects in polarized deposition of the cell wall and increased sensitivity to perturbation of cell wall synthesis. Our results reveal that the NPFX(1,2)D-Sla1p system is responsible for directing Wsc1p into an endocytosis and recycling pathway necessary to maintain yeast cell wall polarity. The dynamic localization of Wsc1p, a sensor of the extracellular wall in yeast, resembles polarized distribution of certain extracellular matrix-sensing integrins through endocytic recycling.     

Specific sterols required for the internalization step of endocytosis in yeast

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.     

Actin and fimbrin are required for the internalization step of endocytosis in yeast.

In Saccharomyces cerevisiae, alpha-factor is internalized by receptor-mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta-tubulin and actin mutant strains, we showed that actin plays a direct role in receptor-mediated internalization of alpha-factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta-tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin-binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha-factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.     

ATP is required for receptor-mediated endocytosis in intact cells

We have demonstrated a requirement for cellular ATP in the receptor- mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric- transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I- BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.     

Hedgehog signaling via angiopoietin1 is required for developmental vascular stability

The molecular pathways by which newly formed, immature endothelial cell tubes remodel to form a mature network of vessels supported by perivascular mural cells are not well understood. The zebrafish iguana (igu) genetic mutant has a mutation in the daz-interacting protein 1 (dzip1), a member of the hedgehog signaling pathway. Loss of dzip1 results in decreased size of the cranial dorsal aortae, ultrastructural defects in perivascular mural cell recruitment and subsequent hemorrhage. Although hedgehog signaling is disrupted in igu mutants, we find no defects in vessel patterning or artery–vein specification. Rather, we show that the loss of angiopoietin1 (angpt1) expression in ventral perivascular mesenchyme is responsible for vascular instability in igu mutants. Over-expression of angpt1 or partial down-regulation of the endogenous Angpt1 antagonist angpt2 rescues hemorrhage. This is the first direct in vivo link between hedgehog signaling and the induction of vascular stability by recruitment of perivascular mural cells through angiopoietin signaling.     

Fine-tuning activity-dependent bulk endocytosis via kinases and phosphatases

The regulation of activity-dependent bulk endocytosis, the dominant mode of membrane retrieval in response to intense neuronal activity, is poorly understood. In this JCB issue, Peng et al. (2021. J. Cell. Biol. https://doi.org/10.1083/jcb.202011028) propose a novel molecular mechanism for the coordination of activity-dependent bulk endocytosis that builds on Minibrain kinase and its presynaptic substrate synaptojanin-1.

Brain function necessitates sustained synaptic transmission regardless of activity demands. The preservation of synaptic transmission depends on the efficient (re)formation of synaptic vesicles (SVs) by endocytosis after their insertion into the synaptic plasma membrane during neuronal stimulation (1). During mild and sparse stimulation, the dominant endocytosis modes are ultrafast endocytosis and clathrin-mediated endocytosis (CME; 1). Both modes appear to have a fixed rate and limited capacity, and therefore cannot adapt to high frequency stimulations that accumulate inserted SV membranes at the presynaptic terminal. Under these conditions, a different endocytosis mode is predominantly used, termed activity-dependent bulk endocytosis (ADBE). ADBE retrieves large areas of the presynaptic plasma membrane to form bulk endosomes, from which new SVs are then generated (1). This form of endocytosis is particularly common in synapses that operate with high rates of neurotransmission, e.g., ribbon synapses of sensory neurons. ADBE contributes to presynaptic plasticity, having recently been demonstrated to control neurotransmitter release probability (2). Importantly, defects in ADBE and SV endocytosis in general have profound consequences on neuronal function and survival, with dysfunction linked to a series of neurodevelopmental disorders (3).Considering the importance of ADBE to brain physiology and pathology, it is essential to understand the molecular machinery that controls this process and synchronizes it with other synaptic events. Amazingly, despite the fact that ADBE was described in the early 1970s, its regulation remains mysterious. Several protein kinases and phosphatases that contribute to regulation of CME and other endocytosis modes (1) may also contribute to ADBE. For example, the calcium/calmodulin-dependent phosphatase calcineurin activates ADBE, working with glycogen synthase kinase-3 to provide bidirectional control via the phosphorylation of specific substrates (4). However, many presynaptic proteins are calcineurin substrates, suggesting other protein kinases may perform complementary roles.In a recent paper, Chang and colleagues (5) present data in support of calcineurin and Minibrain (Mnb) as coregulators of ADBE in fruit flies via bidirectional control of the phosphorylation status of synaptojanin (Synj)-1 phosphatase. The authors argue that the Synj-1 phosphorylation status coordinates the activity-dependent balance between CME versus ADBE (Fig. 1). Namely, during mild stimulation CME is promoted by Mnb, while ADBE is inhibited. During intense stimulation, dephosphorylation of Synj-1 by calcineurin is required to activate ADBE (Fig. 1). An interesting novel aspect arises from examination of domain-specific Synj-1 mutants: its 4′-phosphatase SAC1 activity supports ADBE, while its 5′-phosphatase (5′-PPase) domain suppresses it. The Bin/Amphiphysin/Rvs domain protein endophilin-A has been implicated in ADBE (6); however, a Synj-1 mutant lacking the endophilin-A binding proline-rich domain (PRD) had no effect. Further studies may therefore be required to dissect synaptojanin-1–dependent and –independent roles of endophilin in ADBE.Open in a separate windowFigure 1.Control of CME and ADBE via Minibrain kinase and calcineurin phosphatase. Synj-1 is phosphorylated by Mnb kinase on Ser1029 on its PRD. This promotes the 5′-PPase activity of Synj-1 and inhibits association with the endocytosis protein endophilin-A. These events promote CME. During intense neuronal activity, calcineurin (CaN) is activated and dephosphorylates Synj-1. This reduces 5′-PPase activity and promotes association with endophilin. The dephosphorylation also promotes ADBE via inhibition of Synj-1 5′-PPase activity. This phospho-regulation of the endophilin interaction does not impact ADBE. The SAC activity of Synj-1 is essential for ADBE and is unaffected by phosphorylation.Collectively, the data by Chang and colleagues consolidate the key role played by calcineurin in ADBE and identify Mnb as a new ADBE protein kinase. Intriguingly, the number of synapses performing ADBE is increased in Mnb hypomorphs, suggesting there is additional endocytic capacity that can be recruited on demand. There also appears to be bidirectional control of ADBE via Mnb, since Mnb overexpression represses this pathway. Notably, the enzyme activities of Synj-1 are regulated by Mnb- and calcineurin-dependent turnover of phosphorylation of S1029 (Fig. 1; 7, 8). In mammals, cyclin-dependent kinase 5 is suggested to control Synj-1 activity (9); therefore, it important to confirm whether Synj-1 is also phosphorylated by the Mnb orthologue, dual specificity tyrosine-phosphorylation-regulated kinase (DYRK1A), in mammals. A key test of the causality of activity-dependent phosphorylation events is whether they occur to the same stimulation intensities as the biological event. In this study, activity-dependent dephosphorylation of S1029 on Synj-1 was not demonstrated; instead, an absence of activity-dependent Mnb phosphorylation was observed. In mitigation, the authors convincingly demonstrated that Synj-1 phosphorylation increased during prolonged stimulus in the absence of calcineurin function.This work also confirmed a key role for the phospholipid PI(4,5)P₂ in ADBE (1). Interestingly, it further revealed a hitherto undiscovered role for the SAC domain, but not the 5′-PPase domain of Synj-1 in ADBE. This latter activity is essential for other forms of endocytosis, such as CME and ultrafast endocytosis, with SAC activity required for clathrin-dependent vesicle generation from endosomes (10, 11). In addition to potential roles for Synj-1 SAC activity discussed by Chang and colleagues, a more provocative (and simplistic) explanation is that the end product, phosphatidylinositol (PI) itself, is important for ADBE. In support, the neurons without diacylglycerol kinase (which generates the PI precursor phosphatidic acid) display SV endocytosis defects that are exacerbated during high activity (12).A lack of accurate assays that monitor ADBE in both time and space has limited research in small nerve terminals for decades. In this work, ADBE is evoked and monitored using multiple approaches. This is important, since there is no simple method to monitor ADBE; therefore, it requires cross corroboration wherever possible. This study was greatly assisted via the use of genetically tractable model organisms, allowing precise intervention to abate the function of key proteins and enzymes in vivo. Yet, the trade-off is the relative imprecision of stimulation to evoke SV turnover, with prolonged periods of stimulation (and parallel inhibition of CME) required to evoke and isolate ADBE.Since Peng et al. shed light on new aspects of ADBE regulation, further questions can now be envisioned. In particular, how localized production and degradation of membrane phospholipids coordinate the temporal and spatial triggering of specific endocytosis modes. The essential role for calcineurin in most forms of endocytosis suggests where and when dephosphorylation events occur at the presynapse may be critical in the recruitment of discrete SV reformation pathways. Furthermore, Mnb/DYRK1A is linked to brain pathologies, including Down’s syndrome and autism-spectrum disorders, which is yet to be explored. These and other questions will no doubt drive further studies of remarkable plasticity when it comes to formation of new SVs and synaptic transmission, and how they organize and govern our brain activity.     

Casein kinase II activity is required for transferrin receptor endocytosis.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.     

Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCgamma binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCgamma as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.     

Sphingoid base metabolism in yeast: mapping gene expression patterns into qualitative metabolite time course predictions

Can qualitative metabolite time course predictions be inferred from measured mRNA expression patterns? Speaking against this possibility is the large number of ‘decoupling’ control points that lie between these variables, i.e. translation, protein degradation, enzyme inhibition and enzyme activation. Speaking for it is the notion that these control points might be coordinately regulated such that action exerted on the mRNA level is informative of action exerted on the protein and metabolite levels. A simple kinetic model of sphingoid base metabolism in yeast is postulated. When the enzyme activities in this model are modulated proportional to mRNA expression levels measured in heat shocked yeast, the model yields a transient rise and fall in sphingoid bases followed by a permanent rise in ceramide. This finding is in qualitative agreement with experiments and is thus consistent with the aforementioned coordinated control system hypothesis.     

Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors

The Saccharomyces cerevisiae a-factor receptor (STE3) is subject to two modes of endocytosis: a constitutive process that occurs in the absence of ligand and a regulated process that is triggered by binding of ligand. Both processes result in delivery of the receptor to the vacuole for degradation. Receptor mutants deleted for part of the COOH- terminal cytoplasmic domain are disabled for constitutive, but not ligand-dependent internalization. Trans-acting mutants that impair constitutive endocytosis have been isolated. One of these, ren1-1, is blocked at a late step in the endocytic pathway, as receptor accumulates in a prevacuolar endosome-like compartment. REN1 is identical to VPS2, a gene required for delivery of newly synthesized vacuolar enzymes to the vacuole. Based on this identity, we suggest a model in which the transport pathways to the vacuole--the endocytic pathway and the vacuolar biogenesis pathway--merge at an intermediate endocytic compartment. As receptor also accumulates at the surface of ren1 cells, receptor may recycle from the putative endosome to the surface, or REN1 may also be required to carry out an early step in endocytosis.     

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. We have identified loss-of-function mutations in dshc, the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain completely abolishes DSHC function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, DSHC is involved in signaling by only a subset of RTKs: dshc mutants show defects in Torso and DER but not Sevenless signaling, which is confirmed by epistasis experiments. We show by double-mutant analysis that the adaptors DOS, DRK, and DSHC act in parallel to transduce the Torso signal. Our results suggest that DSHC confers specificity to receptor signaling.     

Calcium-independent calmodulin requirement for endocytosis in yeast.

We have recently shown that actin and fimbrin are required for the internalization step of endocytosis in yeast. Using a yeast strain with a temperature-sensitive allele of CMD1, encoding calmodulin, we demonstrate that this protein is also required for this process. Calmodulin mutants that have lost their high-affinity calcium binding sites are, however, able to carry out endocytosis normally. A mutation in Myo2p, an unconventional myosin that is a possible target of calmodulin, did not inhibit endocytosis. The function of calmodulin in endocytosis seems to be specific among membrane trafficking events, because the calmodulin mutants are not defective for biogenesis of soluble vacuolar hydrolases nor invertase secretion. Calmodulin does not seem to play a major role in the post-internalization steps of the endocytic pathway in yeast.     

Hsc70 is required for endocytosis and clathrin function in Drosophila

By screening for Drosophila mutants exhibiting aberrant bride of sevenless (Boss) staining patterns on eye imaginal disc epithelia, we have recovered a point mutation in Hsc70-4, the closest homologue to bovine clathrin uncoating ATPase. Although the mutant allele was lethal, analysis of mutant clones generated by FLP/FRT recombination demonstrated that the Sevenless-mediated internalization of Boss was blocked in mutant Hsc70-4 eye disc epithelial cells. Endocytosis of other probes was also greatly inhibited in larval Garland cells. Immunostaining and EM analysis of the mutant cells revealed disruptions in the organization of endosomal/lysosomal compartments, including a substantial reduction in the number of clathrin-coated structures in Garland cells. The Hsc70-4 mutation also interacted genetically with a dominant-negative mutant of dynamin, a gene required for the budding of clathrin-coated vesicles (CCVs). Consistent with these phenotypes, recombinant mutant Hsc70 proteins exhibited diminished clathrin uncoating activity in vitro. Together, these data provide genetic support for the long-suspected role of Hsc70 in clathrin-mediated endocytosis, at least in part by inhibiting the uncoating of CCVs.     

The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast.

Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.     

Alix is required for activity-dependent bulk endocytosis at brain synapses

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

The adaptor protein Alix (PDCD6IP) is necessary for membrane shaping underlying various biological processes including endocytosis. This study shows that Alix mediates activity-dependent bulk endocytosis and is required for correct synaptic physiology under normal and pathological conditions.