Visualization of Receptor-mediated Endocytosis in Yeast

The Journal of cell …, 2008

Molecular Biology of the Cell, 2003

Different distribution patterns of the arginine/H+symporter Can1p, the H+plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP– and Pma1p-RFP–containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentati...

PLoS ONE, 2014

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.

PLoS ONE, 2012

In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.

The yeast a -factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a liganddependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a -factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a -factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a -factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.

The Journal of Cell Biology, 1994

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin f...

Febs Letters, 2008

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 livecell 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.

Molecular and Cellular Biology, 1991

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae a-factor, and lIa, the secreted form of P-lactamase encoded by the bla gene of pBR322. The Ste2 and fla components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in ,la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was ceUl associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of DIa; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in Pla secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of (la activity occurred, which is consistent with inversion of the orientation of the Pla reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced (la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of (la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.

Yeast, 2010

The plasma membrane of Saccharomyces cerevisiae contains large microdomains enriched in ergosterol, which house at least nine integral proteins, including proton symporters. The domains adopt a characteristic structure of furrow‐like invaginations typically seen in freeze‐fracture pictures of fungal cells. Being stable for the time comparable with the cell cycle duration, they might be considered as fixed islands (rafts) in an otherwise fluid yeast plasma membrane. Rapidly moving endocytic marker proteins avoid the microdomains; the domain‐accumulated proton symporters consequently show a reduced rate of substrate‐induced endocytosis and turnover. Copyright © 2010 John Wiley & Sons, Ltd.

2013

Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein-tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.

Journal of cell science, 1998

The internalization step of endocytosis has been the focus of several laboratories during the last forty years. Unlike some other budding events in the cell, many fundamental questions regarding the molecular machinery involved in the mechanism of budding itself still remain unsolved. Over the last few years the general picture of the field has quickly evolved from the originally simplistic view which postulated that clathrin polymerization is the major force driving budding at the plasma membrane. Refinement of the assays and molecular markers to measure endocytosis in animal cells has shown that other factors in addition to the clathrin coat are required and that endocytosis can also take place through clathrin-independent mechanisms. At the same time, recent introduction of genetic approaches to study endocytosis has accelerated the identification of molecules required for this process. The isolation of endocytosis mutants in budding yeast has been especially fruitful in this respect. Preliminary comparison of the results obtained in yeast and animal cells did not seem to coincide, but further progress in both systems now suggests that part of the divergence originally seen may be due to the particular experimental approaches used rather than fundamental differences in endocytic mechanisms. In this review we present a short historical overview on the advances made in yeast and animal cells regarding the study of endocytosis, underlining both emerging similarities and still interesting differences.

The Journal of cell biology, 1993

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 accum...

1998

Developmental Cell, 2005

Newly forming clathrin-coated pits accumulate dy-1 Department of Molecular Biology and Microbiology namin and AP-2 in parallel with clathrin. Recruitment or Case Western Reserve University capture of cargo may be important for stabilizing early Cleveland, Ohio 44106 pits so that they continue to grow and complete coated 2 Miami Project to Cure Paralysis vesicle formation (Ehrlich et al., 2004). Also, the size of 3 Department of Molecular and Cellular Pharmacology the cargo influences the size and length of time it takes University of Miami to form a CCV. Coated pits that do not stabilize undergo Miami, Florida 33101 collapse and uncoat without budding. Finally, vesicle release is accompanied by a burst of dynamin recruitment, followed by transient actin polymerization, which Summary is thought to propel vesicles away from the surface (Ehrlich et al., 2004; Merrifield et al., 2002). Additionally, Clathrin-mediated transport is a major pathway for actin is implicated at other stages of endocytosis, inendocytosis. However, in yeast, where cortical actin cluding organization of sites of coated pit formation, patches are essential for endocytosis, plasma memcoated pit assembly, and constriction and/or scission brane-associated clathrin has never been observed. of CCVs (Bennett et al., 2001; Engqvist-Goldstein et al., Using live cell imaging, we demonstrate cortical 2004; Gaidarov et al., 1999; Yarar et al., 2005). clathrin in association with the actin-based endocytic In Saccharomyces cerevisiae, a dynamic actin cytomachinery in yeast. Fluorescently tagged clathrin is skeleton plays a central role in endocytosis (Engqvistfound in highly mobile internal trans-Golgi/endoso-Goldstein and Drubin, 2003). Treatment of yeast with mal structures and in smaller cortical patches. Total actin polymerization inhibitors dramatically blocks ininternal reflection fluorescence microscopy showed ternalization (Ayscough, 2000; Lappalainen and Drubin, that cortical patches are likely endocytic sites, as 1997). In addition, many factors required for proper clathrin is recruited prior to a burst of intensity of the cortical actin organization are also important for enactin patch/endocytic marker, Abp1. Clathrin also acdocytosis and are homologs of proteins involved in cumulates at the cortex with internalizing ␣ factor reclathrin-mediated uptake in other organisms (Engqvistceptor, Ste2p. Cortical clathrin localizes with epsins Goldstein and Drubin, 2003). These include the ep-Ent1/2p and AP180s, and its recruitment to the sursins Ent1p and Ent2p, the EH domain proteins End3p face is dependent upon these adaptors. In contrast, and Eps15-related Pan1p, amphiphysins Rvs161p and Sla2p, End3p, Pan1p, and a dynamic actin cytoskele-Rvs167p, the intersectin-like Sla1p, the Hip1/R homoton are not required for clathrin assembly or exlog Sla2p, and kinases such as Ypk1/2, related to PKB change but are required for the mobility, maturation, and SGK, and actin-regulating kinases Ark1 and Prk1 and/or turnover of clathrin-containing endocytic homologs of animal AAK1 and GAK (Engqvist-Goldstein structures. and Drubin, 2003). Many of these proteins associate transiently with cortical actin patches, which are be-Introduction lieved to form at sites of endocytosis (Engqvist-Goldstein and Drubin, 2003; Huckaba et al., 2004; Jonsdottir and Li, Clathrin is a major vesicle coat protein involved in re-2004; Kaksonen et al., 2003). ceptor-mediated endocytosis (RME) (Brodsky et al., Recent real-time microscopy of fluorescently labeled 2001). Sorting into clathrin-coated pits (CCP) and forendocytic/cortical patch proteins has provided novel mation of clathrin-coated vesicles (CCV) are mediated insight into the dynamics of these factors during endothrough adaptors, which directly bind cargo or bridge cytosis in yeast (Huckaba et al., 2004; Jonsdottir and interactions of clathrin to other endocytic factors and Li, 2004; Kaksonen et al., 2003). Initially Sla1p, Sla2p, inositol phospholipid-containing membrane. The globular Pan1p, and Las17p are recruited to cortical sites. This N-terminal domain (TD) of clathrin heavy chain (HC) is is followed by the assembly of actin, Arp2/3 complexes, important for binding to membrane-associated adapand Abp1. As vesicles/patches appear to pinch off, the tors, including AP-2, AP180/CALM, amphiphysin, epsin, early patch proteins are released and actin/Abp1 Hip1, ARH, and Dab2 (Traub, 2003). Also, actin and actinpatches move rapidly away from the cortex (Kaksonen associated factors play a role in the clathrin-mediated enet al., 2003). This late event is accompanied by recruitdocytosis (Engqvist-Goldstein and Drubin, 2003). ment of type I myosins, Myo3p and Myo5p, which are Recent studies in animal cells using components of hypothesized to function in vesicle fission (Jonsdottir the endocytic machinery tagged with green fluorescent and Li, 2004). Finally, internalized vesicles labeled with protein (GFP) derivatives have allowed examination of the lipophilic dye FM 4-64 and containing Abp1 have the dynamics of clathrin-mediated endocytosis in vivo, been observed moving from the bud cortex into the including measurements of the timing of endocytic mother cell along actin cables (Huckaba et al., 2004). events and the order of machinery and cargo recruit-Though details of the dynamics of endocytosis are emerging from yeast and animal studies, in yeast the role of clathrin in endocytosis remains unclear. Previous

PloS one, 2015

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