A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15

A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.     

Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.     

Cooperative regulation of endocytic vesicle transport by yeast Eps15-like protein Pan1p and epsins

Dynamic actin filaments are required for the formation and internalization of endocytic vesicles. Yeast actin cables serve as a track for the translocation of endocytic vesicles to early endosomes, but the molecular mechanisms regulating the interaction between vesicles and the actin cables remain ambiguous. Previous studies have demonstrated that the yeast Eps15-like protein Pan1p plays an important role in this interaction, and that interaction is not completely lost even after deletion of the Pan1p actin-binding domain, suggesting that additional proteins mediate association of the vesicle with the actin cable. Other candidates for mediating the interaction are endocytic coat proteins Sla2p (yeast Hip1R) and Ent1p/2p (yeast epsins), as these proteins can bind to both the plasma membrane and the actin filament. Here, we investigated the degree of redundancy in the actin-binding activities of Pan1p, Sla2p, and Ent1p/2p involved in the internalization and transport of endocytic vesicles. Expression of the nonphosphorylatable form of Pan1p, Pan1-18TA, caused abnormal accumulation of both actin cables and endocytic vesicles, and this accumulation was additively suppressed by deletion of the actin-binding domains of both Pan1p and Ent1p. Interestingly, deletion of the actin-binding domains of Pan1p and Ent1p in cells lacking the ENT2 gene resulted in severely defective internalization of endocytic vesicles and recruitment of actin cables to the site of endocytosis. These results suggest that Pan1p and Ent1p/2p cooperatively regulate the interaction between the endocytic vesicle and the actin cable.     

The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis

Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70 x 200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.     

Taking apart the endocytic machinery

The formation of clathrin-coated endocytic vesicles is driven by a complex and highly dynamic molecular machinery. In this issue, Idrissi et al. (Idrissi, F.-Z., H. Grötsch, I.M. Fernández-Golbano, C. Presciatto-Baschong, H. Riezman, and M.-I. Geli. 2008. J. Cell Biol. 180:1219–1232) reveal some of the secrets of this machinery by analyzing the localizations of nine endocytic proteins during vesicle budding in yeast using quantitative immunoelectron microscopy.More than 50 different proteins are thought to have roles in the formation of clathrin-coated endocytic vesicles. These proteins assemble together at the plasma membrane, forming the molecular machinery that drives budding of endocytic vesicles. Although clathrin-mediated endocytosis has been studied already for more than four decades, an understanding of the molecular mechanisms of the process is still quite limited. The difficulty of unraveling the molecular mechanisms is not only a result of the large number of involved proteins but is also a result of the dynamic nature of the endocytic machinery. Endocytic proteins are recruited to the site of vesicle formation in a sequential manner, each protein having its specific times of arrival and departure. The composition of the endocytic machinery can change in a matter of seconds. Many of the recent insights into the process of clathrin-mediated endocytosis have come from imaging of fluorescently labeled proteins in living cells using fluorescence microscopy. Light microscopy provides a good temporal resolution of dynamic events, but its spatial resolution is quite limiting when studying endocytic vesicle budding. On the other hand, electron microscopy offers much better spatial resolution but only provides still images.In this issue, one study (see Idrissi et al. on p. 1219) uses immunoelectron microscopy to study the localizations of nine different proteins at sites of endocytosis in yeast cells. Most of the proteins involved in clathrin-mediated endocytosis in yeast are conserved throughout eukaryotes, including mammals, making yeast a good model system for studying the basic mechanisms of endocytosis. However, only a few studies have addressed the organization of the endocytic machinery at the ultrastructural level in yeast (Mulholland et al., 1994; Young et al., 2004; Rodal et al., 2005). Idrissi et al. (2008) start by looking at clathrin, Pan1 (Eps15 homologue), and Sla1 (intersectin-like protein), which, when visualized in living cells by fluorescence microscopy, show similar behaviors. These proteins accumulate at the plasma membrane, forming small fluorescent spots that are initially nonmotile but then move ∼200 nm from the surface toward the interior of the cell at a constant speed for ∼10 s, after which the spots are rapidly disassembled (Kaksonen et al., 2005; Newpher et al., 2005). Idrissi et al. (2008) show by immunoelectron microscopy that clathrin, Pan1, and Sla1 each localize to tips of plasma membrane invaginations, which are ∼50 nm in diameter and have variable lengths up to 180 nm (Fig. 1). This confirms the earlier hypothesis that the movement of these proteins seen in living cells corresponds to the invagination of a clathrin-coated pit, not to the movement of an already budded vesicle. Importantly, these observations show that the length of the endocytic invagination can be used as an indicator for its age.Open in a separate windowFigure 1. Schematic model showing the localization of nine proteins on an endocytic invagination. An invagination of intermediate length (∼100 m) is depicted. The coat proteins, including clathrin, coat the tip of the invagination. Rvs167, Las17, and Bbc1 occupy the neck region below the tip. Myo5 concentrates to the base of the invagination. Actin and actin-binding protein Abp1 form a shell covering the whole invagination. The quantitative immunoelectron microscopy analysis is then applied to six other proteins involved in endocytosis: Rvs167, Las17, Bbc1, Myo5, actin, and Abp1 (Fig. 1). Rvs167 is a homologue of mammalian amphiphysin, a protein involved in pinching vesicles off from the tips of clathrin-coated pits (Takei et al., 1999). The other studied proteins are regulators or components of the actin cytoskeleton, which, in yeast, is essential for endocytosis, specifically for the movement of clathrin and other coat-associated proteins away from the cell surface (i.e., for the membrane invagination; Kubler and Riezman, 1993; Kaksonen et al., 2003). This analysis reveals many exciting details about the dynamic organization of the endocytic machinery. The yeast amphiphysin homologue Rvs167 is shown to localize to the tubular area of the membrane invagination, just below its clathrin-coated tip. Las17 (yeast Wiskott-Aldrich syndrome protein), a strong activator of the actin filament nucleator Arp2/3 (Winter et al., 1999), and Bbc1, an inhibitor of Las17 (Rodal et al., 2003), both localize to the same area as Rvs167. Myo5, a type I myosin, which is both an actin-dependent molecular motor and an activator of the Arp2/3 complex (Sun et al., 2006), localizes mostly to the base of the invagination, where the membrane has a negative curvature. Genetic experiments together with live cell imaging have suggested that Las17 and Myo5 are both needed sequentially for actin-driven invagination of the membrane (Sirotkin et al., 2005; Sun et al., 2006; Galletta et al., 2008). Las17 has a role in initiating the actin polymerization at endocytic sites, whereas Myo5 is needed for the subsequent internalization process. Interestingly, these two major activators of the Arp2/3 complex localize slightly differently: Myo5 closer to the base of the invagination and Las17 in the middle. This suggests that actin polymerization may be spatially restricted to different areas during different stages of endocytosis. Analysis of actin and actin filament–binding protein Abp1 reveals that they are localized throughout the invagination. However, when compared with the other proteins, immunogold labeling for actin and Abp1 is significantly further away from the lipid bilayer, suggesting that the actin cytoskeleton forms an outer shell covering the rest of the endocytic protein machinery.Using the invagination length as an indicator for the age of the endocytic site, Idrissi et al. (2008) are able to add the time dimension to their data, revealing some interesting temporal dynamics of protein localizations. The temporal order of protein recruitment derived from the electron microscopy data matches observations made using live cell imaging, but the localizations can now be seen at much higher resolution and in relation to the shape of the membrane. Bbc1 and Rvs167 colocalize with Las17, but they appear only on longer, older invaginations. Similarly, Las17 localization precedes Myo5 accumulation, which is consistent with their postulated order of function. The shortest invaginations (<50 nm) show very little labeling for actin. This may mean that the initial membrane bending is independent of actin and could be caused by clathrin or other coat proteins. In older invaginations, actin shows an intriguing distribution. The initial continuous labeling is split into two. Part of the staining localizes to the base of the invagination, and another part localizes to the tip. Similar behavior is also described for Myo5, which initially is concentrated at the base of the invagination but later also appears at the tip. It is not clear whether this staining pattern reflects two separate structures or whether one structure breaks into two. However, this finding shows that the organization of the actin cytoskeleton associated with the endocytic sites may be more complex than previously thought.One of the key events on the endocytic pathway, vesicle scission, still escapes analysis. Scission and the following disassembly are probably too transient to be caught in fixed cells frequently enough to yield sufficient data for analysis. Other very transient events may also go undetected because they could get smeared as a result of the averaging of data from tens of different invaginations. For these very transient events, live cell imaging is likely to remain the method of choice (Merrifield et al., 2005). However, the superior resolution offered by electron microscopy will clearly continue to provide critical insights. Idrissi et al. (2008) analyzed localizations of nine different proteins. At least 40 yeast proteins involved in endocytic internalization remain to be studied. The rich collections of endocytic mutants will also provide many interesting samples for analysis. What happens to the organization of the endocytic machinery when one of the Arp2/3 activators is mutated? Does the shape or size of the invagination change if one of the coat components is deleted? What would be the effect of inhibiting the motor activity of Myo5? These are just a few examples of exciting questions that can now be addressed.     

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.     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

Cortactin is a component of clathrin-coated pits and participates in receptor-mediated endocytosis

The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

A Highlights from MBoC Selection: Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30–50% between individual patches. The pathway begins with accumulation of 30–40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (∼120 End4p and ∼230 Pan1p), activators of Arp2/3 complex (∼200 Wsp1p and ∼340 Myo1p) and ∼300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (∼7000 actins, ∼200 capping proteins, and ∼900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over ∼10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803–2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100–200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.     

Clathrin light chain directs endocytosis by influencing the binding of the yeast Hip1R homologue, Sla2, to F-actin

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin-binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the clathrin lattice, providing direction for membrane internalization.     

Defects in structural integrity of ergosterol and the Cdc50p-Drs2p putative phospholipid translocase cause accumulation of endocytic membranes, onto which actin patches are assembled in yeast

Specific changes in membrane lipid composition are implicated in actin cytoskeletal organization, vesicle formation, and control of cell polarity. Cdc50p, a membrane protein in the endosomal/trans-Golgi network compartments, is a noncatalytic subunit of Drs2p, which is implicated in translocation of phospholipids across lipid bilayers. We found that the cdc50Delta mutation is synthetically lethal with mutations affecting the late steps of ergosterol synthesis (erg2 to erg6). Defects in cell polarity and actin organization were observed in the cdc50Delta erg3Delta mutant. In particular, actin patches, which are normally found at cortical sites, were assembled intracellularly along with their assembly factors, including Las17p, Abp1p, and Sla2p. The exocytic SNARE Snc1p, which is recycled by an endocytic route, was also intracellularly accumulated, and inhibition of endocytic internalization suppressed the cytoplasmic accumulation of both Las17p and Snc1p. Simultaneous loss of both phospholipid asymmetry and sterol structural integrity could lead to accumulation of endocytic intermediates capable of initiating assembly of actin patches in the cytoplasm.     

SLAC,a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how Las17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that Las17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits Las17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on the class I/II Las17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to Las17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.     

The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p

A screen for mutations that affect the recruitment of the exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the exocyst with clathrin was seen, arguing against a direct role in exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the exocyst. We observe a direct interaction between the exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.     

A pathway for association of receptors, adaptors, and actin during endocytic internalization

In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.     

Functional genomic screen identifies novel mediators of collagen uptake

Tissue fibrosis occurs when matrix production outpaces matrix degradation. Degradation of collagen, the main component of fibrotic tissue, is mediated through an extracellular proteolytic pathway and intracellular pathway of cellular uptake and lysosomal digestion. Recent studies demonstrate that disruption of the intracellular pathways can exacerbate fibrosis. These pathways are poorly characterized. Here we identify novel mediators of the intracellular pathway of collagen turnover through a genome-wide RNA interference screen in Drosophila S2 cells. Screening of 7505 Drosophila genes conserved among metazoans identified 22 genes that were required for efficient internalization of type I collagen. These included proteins involved in vesicle transport, the actin cytoskeleton, and signal transduction. We show further that the flotillin genes have a conserved and central role in collagen uptake in Drosophila and human cells. Short hairpin RNA–mediated silencing of flotillins in human monocyte and fibroblasts impaired collagen uptake by promoting lysosomal degradation of the endocytic collagen receptors uPARAP/Endo180 and mannose receptor. These data provide an initial characterization of intracellular pathways of collagen turnover and identify the flotillin genes as critical regulators of this process. A better understanding of these pathways may lead to novel therapies that reduce fibrosis by increasing collagen turnover.     

Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475-487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission.     

Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast

Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

Proteomic Analysis of Clathrin Interactions in Trypanosomes Reveals Dynamic Evolution of Endocytosis

Endocytosis is a vital cellular process maintaining the cell surface, modulating signal transduction and facilitating nutrient acquisition. In metazoa, multiple endocytic modes are recognized, but for many unicellular organisms the process is likely dominated by the ancient clathrin‐mediated pathway. The endocytic system of the highly divergent trypanosomatid Trypanosoma brucei exhibits many unusual features, including a restricted site of internalization, dominance of the plasma membrane by GPI‐anchored proteins, absence of the AP2 complex and an exceptionally high rate. Here we asked if the proteins subtending clathrin trafficking in trypanosomes are exclusively related to those of higher eukaryotes or if novel, potentially taxon‐specific proteins operate. Co‐immunoprecipitation identified twelve T. brucei clathrin‐associating proteins (TbCAPs), which partially colocalized with clathrin. Critically, eight TbCAPs are restricted to trypanosomatid genomes and all of these are required for robust cell proliferation. A subset, TbCAP100, TbCAP116, TbCAP161 and TbCAP334, were implicated in distinct endocytic steps by detailed analysis of knockdown cells. Coupled with the absence of orthologs for many metazoan and fungal endocytic factors, these data suggest that clathrin interactions in trypanosomes are highly lineage‐specific, and indicate substantial evolutionary diversity within clathrin‐mediated endocytosis mechanisms across the eukaryotes.