A Yeast t-SNARE Involved in Endocytosis

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Δ cells, internalization was normal for two endocytic markers, the pheromone α-factor and the plasma membrane uracil permease. In contrast, degradation of α-factor and uracil permease was delayed in tlg2Δ cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.     

Ordering of compartments in the yeast endocytic pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t-SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t-SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.     

Evidence for two endocytic transport pathways in plant cells

Intracellular trafficking of endocytic vesicles in eukaryotes varies with the nature of the cargo molecules and the targeted organelle, and proceeds through an intricate network of internal endosomal compartments. However, the path for fluid-phase endocytosis (FPE), the internalization of external solutes from the apoplast via plasmalemma generated vesicles, remains unresolved despite some indication of a direct transport route to the vacuole. To test this hypothesis, we made use of the membrane-impermeable Na-dependent fluorescent marker Coro-Na in combination with the fluorescent membrane marker FM 4-64 and confocal laser scanning microscopy. When protoplasts from sweet lime juice cells were incubated in Na-free solution, FM 4-64, Coro-Na, and 200 mM sucrose, two distinct types of labeled vesicles were evident. A set of vesicles (1 μm in diameter) was intensely labeled with Coro-Na and to a lesser extent with FM 4-64, whereas the second type of 1–7 μm structures appeared exclusively labeled with FM 4-64. These data demonstrate the parallel functioning of two endocytic pathways in plant cells. In one system, a set of small endocytic vesicles merge with the endosome, whereas a separate set of vesicles fuse to form larger vesicles independent from the endosome. Although it is likely that both vesicle systems eventually contribute to solutes reaching the vacuole, given their size (1–7 μm), and based on previous observations of endocytic vesicle formation protruding from the plasmalemma and merging with the vacuole, we conclude that these latter vesicles constitute the primary FPE vesicle system.     

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids

Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.     

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.     

A WASp homolog powers actin polymerization-dependent motility of endosomes in vivo

BACKGROUND: WASp/SCAR proteins activate the Arp2/3 complex to nucleate actin filament assembly and are thought to have important roles in endocytosis. WASp is required for efficient endocytosis of antigen receptors, N-WASp promotes actin polymerization-dependent movement of endomembrane vesicles, and Las17 (a yeast WASp homolog) is required for endocytic internalization. However, it is unknown whether movement of endosomes or other organelles requires activation of the Arp2/3 complex by members of the WASp/SCAR family. RESULTS: Fluorescence video microscopy of yeast cells expressing a GFP-tagged G protein-coupled receptor (Ste2-GFP) as an endocytic marker revealed that endosomes and the lysosome-like vacuole are highly motile. Endosome/vacuole motility required actin polymerization, as indicated by sensitivity to latrunculin A, whereas microtubules were uninvolved. Endosome/vacuole motility did not require actin cables or myosin V (a MYO2 gene product), which moves secretory vesicles and the Golgi apparatus and mediates vacuole segregation. However, endosome motility required Las17, a WASp homolog. In contrast to other processes involving Las17, endosome/vacuole motility required the WCA domain of Las17, which is necessary and sufficient to activate the Arp2/3 complex. CONCLUSIONS: Endosome/vacuole motility in vivo requires actin polymerization stimulated by the WASp homolog Las17. WASp/SCAR family members in mammalian cells may have similar functions. Defects in endosome/lysosome motility may contribute to deficits in lymphocyte or macrophage function observed in human patients lacking WASp or developmental defects in N-WASp-deficient mice.     

Novel Genes Involved in Endosomal Traffic in Yeast Revealed by Suppression of a Targeting-defective Plasma Membrane ATPase Mutant

A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system. We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome. Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1^ts. 16 independent suppressor of pma1 (sop) mutants were isolated. Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins. A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y. Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants. By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants. Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-α factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2. One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling). Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.     

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.     

Actin-based motility during endocytosis in budding yeast

Actin assembly nucleated by Arp2/3 complex has been implicated in the formation and movement of endocytic vesicles. The dendritic nucleation model has been proposed to account for Arp2/3-mediated actin assembly and movement. Here, we explored the model by examining the role of capping protein in vivo, with quantitative tracking analysis of fluorescence markers for different stages of endocytosis in yeast. Capping protein was most important for the initial movement of endocytic vesicles away from the plasma membrane, which presumably corresponds to vesicle scission and release. The next phase of endosome movement away from the plasma membrane was also affected, but less so. The results are consistent with the dendritic nucleation model's prediction of capping protein as important for efficient actin assembly and force production. In contrast, the movement of late-stage endocytic vesicles, traveling through the cytoplasm en route to the vacuole, did not depend on capping protein. The movement of these vesicles was found previously to depend on Lsb6, a WASp interactor, whereas Lsb6 was found here to be dispensable for early endosome movement. Thus, the molecular requirements for Arp2/3-based actin assembly differ in early versus later stages of endocytosis. Finally, acute loss of actin cables led to increased patch motility.     

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.     

A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis

The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.     

PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P₂], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P₂ production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P₂ is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.     

A Vps21 endocytic module regulates autophagy

In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.     

A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum‐derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system‐mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans‐Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase‐activating Protein (GAP) ACAP1 to dysregulate Arf6‐/Rab8a‐dependent transport within the recycling endosome, which resulted in accretion of TGN‐associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.     

A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae

The late endosome and vacuole of yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome. The late endosome is the convergence point of the biosynthetic and endocytic trafficking to the vacuole. Here, we describe a novel immunodetection screen to isolate mutants defective in trafficking the soluble hydrolase carboxypeptidase Y (CPY) at the late endosome to vacuole interface (env mutants). Mutants exhibit vacuolar morphology and endocytosis defects as assayed by electron, fluorescent, and nomarski microscopy. In biochemical assays, they internally accumulate p2CPY in a dense membrane compartment lacking vacuolar properties yet display normal secretion phenotypes. The results suggest vacuolar morphology and function defects that are exclusively at the late endosome/vacuole interface. env mutants define five complementation groups. The first gene of the collection to be cloned, ENV1 is allelic to VPS35 whose established function is in retrograde trafficking from late endosome to trans-Golgi network (TGN). Microscopic, biochemical, and growth analyses establish that env1 is distinct from other alleles of VPS35 in vacuolar morphology, growth characteristics, and internal accumulation of p2CPY. Our results indicate that ENV genes may define new gene functions at the late endosome to vacuole interface.     

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.     

Candida albicans ABG1 gene is involved in endocytosis

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.     

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal ¹²⁵I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect ¹²⁵I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when ¹²⁵I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired ¹²⁵I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.     

A model of actin-driven endocytosis explains differences of endocytic motility in budding and fission yeast

A comparative study (Sun et al., 2019) showed that the abundance of proteins at sites of endocytosis in fission and budding yeast is more similar in the two species than previously thought, yet membrane invaginations in fission yeast elongate twofold faster and are nearly twice as long as in budding yeast. Here we use a three-dimensional model of a motile endocytic invagination (Nickaeen et al., 2019) to investigate factors affecting elongation of the invaginations. We found that differences in turgor pressure in the two yeast species can largely explain the paradoxical differences observed experimentally in endocytic motility.     

Actin in the endocytic pathway: from yeast to mammals

Genetic analysis of endocytosis in yeast early pointed to the essential role of actin in the uptake step. Efforts to identify the machinery involved demonstrated the important contribution of Arp2/3 and the myosins-I. Analysis of the process using live-cell fluorescence microscopy and electron microscopy have recently contributed to refine molecular models explaining clathrin and actin-dependent endocytic uptake. Increasing evidence now also indicates that actin plays important roles in post-internalization events along the endocytic pathway in yeast, including transport of vesicles, motility of endosomes and vacuole fusion. This review describes the present knowledge state on the roles of actin in endocytosis in yeast and points to similarities and differences with analogous processes in mammals.