Actin and Endocytosis in Budding Yeast

Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed.     

Engineering expression of the heavy metal transporter MerC in Saccharomyces cerevisiae for increased cadmium accumulation

The merC gene from the Tn21-encoded mer operon has potential uses as a molecular tool for bioremediation. It was overexpressed as the fusion proteins MerC-Sso1p or MerC-Vam3p in Saccharomyces cerevisiae. Green fluorescent protein (GFP)-MerC-Sso1p fusion proteins located primarily in the plasma membrane, although some protein was detected in the endoplasmic reticulum. In contrast, GFP-MerC-Vam3p was expressed in the vacuolar membranes. These results suggest that yeast Sso1p and Vam3p are essential for targeting molecules to the plasma and vacuolar membranes, respectively. Significantly more cadmium ions were accumulated by yeast cells expressing MerC-Sso1p than with MerC-Vam3p or control cells. These results suggest that expression of MerC in the plasma membrane may be a particularly promising strategy for improving accumulation of cadmium in yeast.     

Ionophores and intact cells I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae

Valinomycin and nigericin prevented growth of 13 strains of the yeast Saccharomyces cerevisiae on non-fermentable substrate glycerol without affecting much fermentative growth on glucose. The two antibiotics did not induce swelling and lysis of yeast protoplasts in potassium acetate and did not modify uptake and release of Rb⁺ by the yeast cells. Both antibiotics were taken up by yeast cells at a relatively low rate. Nigericin accelerated the glucose-induced changes of fluorescence of a cyanine dye absorbed by yeast cells, which had been previously ascribed to a depolarization-repolarization cycle of the mitochondrial membrane. The data suggest that valinomycin and nigericin act as ionophores in the inner mitochondrial membrane and not in the plasma membrane of intact yeast cells.     

Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H⁺ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [³H]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of d -glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-β-d -glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni²⁺-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.     

Ionophores and intact cells II. Oleficin acts on mitochondria and induces disintegration of the mitochondrial genome in yeast Saccharomyces cerevisiae

The non-macrolid polyene antibiotic oleficin, which has been shown to function as an ionophore of Mg²⁺ in isolated rat liver mitochondria, preferentially inhibited growth of the yeast Saccharomyces cerevisiae on non-fermentable substrates. It uncoupled and inhibited respiration of intact cells and converted both growing and resting cells into respiration-deficient mutants. The mutants arose as a result of fragmentation of the mitochondrial genome. Another antibiotic known to be an ionophore of divalent cations, A23187, also selectively inhibited growth of the yeast on non-fermentable substrates, but did not produce the respiration-deficient mutants, neither antibiotic inhibited the energy-dependent uptake of divalent cations by yeast cells nor opened the plasma membrane for these cations. The results indicate that in Saccharomyces cerevisiae both oleficin and A23187 preferentially affected the mitochondrial membrane without acting as ionophores in the plasma membrane.     

Summary

The plasma membrane H⁺-ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C-terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H⁺-ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H⁺-ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14–3-3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14–3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14–3-3 protein binding motif in Raf-1. The results demonstrate that the 14–3-3 protein is an activator molecule of the H⁺-ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H⁺-ATPase.     

Purification of Functionally Sealed Cytoplasmic Side-out Plasma Membrane Vesicles from Saccharomyces cerevisiae

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 μM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H⁺-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Mechanism of liponecrosis,a distinct mode of programmed cell death

An exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA) elicits “liponecrosis," a mode of programmed cell death (PCD) which differs from the currently known PCD subroutines. Here, we report the following mechanism for liponecrotic PCD. Exogenously added POA is incorporated into POA-containing phospholipids that then amass in the endoplasmic reticulum membrane, mitochondrial membranes and the plasma membrane. The buildup of the POA-containing phospholipids in the plasma membrane reduces the level of phosphatidylethanolamine in its extracellular leaflet, thereby increasing plasma membrane permeability for small molecules and committing yeast to liponecrotic PCD. The excessive accumulation of POA-containing phospholipids in mitochondrial membranes impairs mitochondrial functionality and causes the excessive production of reactive oxygen species in mitochondria. The resulting rise in cellular reactive oxygen species above a critical level contributes to the commitment of yeast to liponecrotic PCD by: (1) oxidatively damaging numerous cellular organelles, thereby triggering their massive macroautophagic degradation; and (2) oxidatively damaging various cellular proteins, thus impairing cellular proteostasis. Several cellular processes in yeast exposed to POA can protect cells from liponecrosis. They include: (1) POA oxidation in peroxisomes, which reduces the flow of POA into phospholipid synthesis pathways; (2) POA incorporation into neutral lipids, which prevents the excessive accumulation of POA-containing phospholipids in cellular membranes; (3) mitophagy, a selective macroautophagic degradation of dysfunctional mitochondria, which sustains a population of functional mitochondria needed for POA incorporation into neutral lipids; and (4) a degradation of damaged, dysfunctional and aggregated cytosolic proteins, which enables the maintenance of cellular proteostasis.     

Sugar transport and potassium permeability in yeast plasma membrane vesicles

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained.The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal.The yield of vesicles sealed to K⁺ was maximal at pH 4 and accounted for about one third of the total vesicle volume.The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K⁺.As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO₂²⁺.Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane.After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.     

An investigation into the feasibility of using yeast protoplasts to study the ion transport properties of the plasma membrane

A method for studying ion uptake in enzymatically isolated protoplasts from the yeast, Saccharomyces cerevisiae, is described. The kinetics of K⁺ and Rb⁺ uptake, metabolic proton extrusion and cell electrophoretic mobility bave been determined. Enzymic removal of the cell wall does not significantly alter the above-mentioned properties of the yeast cells. It is concluded that studies of these properties can be performed equally well with intact yeast cells or protoplasts. However, in studies aimed at determining effects of complex organic substances, e.g., antibiotics, on plasma membrane function the use of protoplasts is recommended. The effectiveness of the antibiotic, Dio-9, for example, in reversing the metabolic proton extrusion into a net proton influx is at least 50 times higher after enzymic removal of the yeast cell wall.     

Functional patchworking at the plasma membrane

Lipids and proteins are not evenly distributed within the plasma membrane (PM), but instead segregate laterally into many specialized microdomains whose functional relevance is not clear. In this issue, Busto et al ( 2018 ) demonstrate that substrate flux through a nutrient transporter drives the lateral relocation of the transporter between specific microdomains at the yeast PM, suggesting that regulating the lateral plasma membrane compartmentalization for individual proteins could be a general process for cellular response to environmental conditions.     

Permeabilization of plant cells for release of intracellularly stored products: viability studies

Summary The effects of various chemical substances on the permeability of plasma membranes and tonoplasts of three suspension cultures (Catharanthus roseus, Thalictrum rugosum and Chenopodium rubrum) have been studied. The permeability of the plasma membrane is monitored by measuring the activity of the cytosolic enzyme isocitrate dehydrogenase and the permeability of the tonoplast is measured by determining the release of substances stored in the vacuoles (inorganic phosphate, berberine and betanin for the three cell lines, respectively). The minimum concentration required for quantitative release of vacuolar products have been established for five different permeabilization agents. Cell viability is lost upon permeabilization except for treatment of Catharanthus roseus with DMSO and Triton X-100.Abbreviations DMSO dimethylsulfoxide - PEA phenethylalcohol - HDTMAB hexadecyltrimethylammonium bromide - ICDH isocitrate dehydrogenase     

Molecular characterization of a cold-induced plasma membrane protein gene from wheat

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Δpmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat. The nucleotide sequence data for wpi6 have been recorded in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AB030210 (cDNA) and AB221353 (genomic DNA).     

Active trans-plasma membrane water cycling in yeast is revealed by NMR

Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal ¹H₂O NMR relaxation time constant (T₁) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τ_i), where τ_i⁻¹ is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O₂/5%CO₂ (O₂) or 95%N₂/5%CO₂ (N₂). ATP was high during O₂, and τ_i⁻¹ was 3.1 s⁻¹ at 25°C. After changing to N₂, ATP decreased and τ_i⁻¹ was 1.8 s⁻¹. The principal active yeast ion transport protein is the plasma membrane H⁺-ATPase. Studies using the H⁺-ATPase inhibitor ebselen or a yeast genetic strain with reduced H⁺-ATPase found reduced τ_i⁻¹, notwithstanding high ATP. Steady-state water exchange correlates with H⁺-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity.     

Effect of proteases,phospholipases and polysaccharide-splitting enzymes on plasma membrane particles and on the synthesis of the fibrillar cell wall component in yeast protoplasts

Plasma membrane particles demonstrable by the freeze-etching technique, play, according to some authors, a role in the cell wall synthesis. On a model of yeast protoplast capable of regenerating the cell wall we studied the morphology of plasma membrane particles and the synthesis of the fibrillar cell wall component following a treatment with various enzymes and with lysolecithin. The enzymes used included proteases (trypsin, papain, pronase), polysaccharide-splitting enzymes (snail enzyme complex, mannosidase), phospholipases (A, C, D) and lipase. Upon treating living protoplasts with these substances in no case did we observe any morphologically demonstrable change in the particle structure or in their distribution in the plasma membrane. The fibrillar cell wall component was synthetized even in the presence of proteases and phospholipases. If the plasma membrane particles are assumed to represent enzyme systems synthesizing the cell wall component then in living protoplasts they are not located on the outer plasma membrane face or else are protected by some mechanism against the action of the corresponding enzymes.     

Anhydrobiosis in yeast: Stabilization by exogenous lactose

We have found that incubation in lactose solutions (0.75 M) of yeast culture Saccharomyces cerevisiae sensitive to dehydration damage increased the stability of the cells during dehydration. Simultaneously with this increase in viability, a decrease in plasma membrane permeability during rehydration was seen. Using Fourier transform infrared spectroscopy to measure lipid phase transitions, we observed that the lactose treatment depressed the membrane phospholipid phase transition temperature in a sensitive culture of dry yeast. As a result, it leads to the decrease in the damages of molecular organization of membranes during rehydration of dry yeast cells, thus reducing leakage from the cells.     

Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation

Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N‐terminal domain homologous to the crescent‐shaped membrane‐tubulating EFC/F‐BAR domains and a C‐terminal domain homologous to cargo‐binding μ homology domains (μHDs). In vitro and in vivo assays confirmed membrane‐tubulation activity for muniscin EFC/F‐BAR domains. The μHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane‐tubulation activity that is important for regulating endocytosis.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.