New Insight Into the Roles of Membrane Microdomains in Physiological Activities of Fungal Cells.

The organization of biological membranes into structurally and functionally distinct lateral microdomains is generally accepted. From bacteria to mammals, laterally compartmentalized membranes seem to be a vital attribute of life. The crucial fraction of our current knowledge about the membrane microdomains has been gained from studies on fungi. In this review we summarize the evidence of the microdomain organization of membranes from fungal cells, with accent on their enormous diversity in composition, temporal dynamics, modes of formation, and recognized engagement in the cell physiology. A special emphasis is laid on the fact that in addition to their other biological functions, membrane microdomains also mediate the communication among different membranes within a eukaryotic cell and coordinate their functions. Involvement of fungal membrane microdomains in stress sensing, regulation of lipid homeostasis, and cell differentiation is discussed more in detail.

Plants and fungi in the era of heterogeneous plasma membranes.

Examples from yeast and plant cells are described that show that their plasma membrane is laterally compartmented. Distinct lateral domains encompassing both specific lipids and integral proteins coexist within the plane of the plasma membrane. The compartments are either spatially stable and include distinct sets of proteins, or they are transiently formed to accomplish diverse functions. They are not related to lipid rafts or their clusters, as defined for mammalian cells. This review summarises only well-documented compartments of plasma membranes from plants and fungi, which have been recognised using microscopic approaches. In several cases, physiological functions of the membrane compartmentation are revealed.

Specific lipid requirements of membrane proteins--a putative bottleneck in heterologous expression.

Membrane proteins are mostly protein-lipid complexes. For more than 30 examples of membrane proteins from prokaryotes, yeast, plant and mammals, the importance of phospholipids and sterols for optimal activity is documented. All crystallized membrane protein complexes show defined lipid-protein contacts. In addition, lipid requirements may also be transitory and necessary only for correct folding and intercellular transport. With respect to specific lipid requirements of membrane proteins, the phospholipid and glycolipid as well as the sterol content of the host cell chosen for heterologous expression should be carefully considered. The lipid composition of bacteria, archaea, yeasts, insects,Xenopus oocytes, and typical plant and mammalian cells are given in this review. A few examples of heterologous expression of membrane proteins, where problems of specific lipid requirements have been noticed or should be thought of, have been chosen.

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H(+)-ATPase reconstituted into vesicles.

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Delta(psi)) generated by the Schizosaccharomyces pombe plasma membrane H(+)-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Delta(psi) because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Delta(psi) at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H(+)-ATPase generates Delta(psi) of about 160 mV on the vesicle membrane. The generated Delta(psi) was stable at least over tens of minutes. An influence of the H(+) membrane permeability on the Delta(psi) buildup was demonstrated by manipulating the H(+) permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H(+)-ATPase-mediated Delta(psi) buildup.

Construction of phosphatidylethanolamine-less strain of Saccharomyces cerevisiae. Effect on amino acid transport.

A triple yeast mutant was constructed which lacks BST1, the gene for sphingosine lyase, besides the phosphatidylserine decarboxylases PSD1 and PSD2. In this yeast mutant, which can only be grown in the presence of exogenous ethanolamine, phosphatidylethanolamine can be depleted to very low levels. Under those conditions, respiration as well as glucose and 3-O-methylglucose uptake proceed unaffected. Plasma membrane ATPase is as active in these cells as that of control cells grown in the presence of ethanolamine. Drastically decreased, however, are H+/amino acid symporters. The activities of arginine (Can1p), proline (Put4p) and general amino acid permease (Gap1p) are decreased more than 20-fold. Amino acid transport in yeast is dependent on coupling to the proton motive force. It can be envisaged that phosphatidylethanolamine might play a role in this process or in the early steps of the secretion pathway common for all amino acid permeases or, eventually, it could affect the transport proteins directly at the plasma membrane Transformation of the triple mutant with a CEN plasmid harbouring BST1 wild-type gene totally reversed its phenotype to that observed in the double mutant.

Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability.

Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 2 l of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family.

The C-terminal tetrapeptide HWFW of the Chlorella HUP1 hexose/H(+)-symporter is essential for full activity and an alpha-helical structure of the C-terminus.

C-terminal tails of plant hexose/H(+)-symporters of the major facilitator superfamily contain a highly conserved motif of four amino acids: HWFW. A deletion of these four amino acids in the Chlorella HUP1 protein leads to a decrease in transport activity by a factor of 3-4. The mutated tail is highly sensitive to trypsin; it does not show alpha-helical conformation in contrast to the wild type C-terminal peptide with an alpha-helical content of at least 15%. The production of monoclonal antibody 416B8 recognizing an epitope within the central loop of HUP1 protein has been a prerequisite for the experiments described.

Expression of eukaryotic plasma membrane transporter HUP1 from Chlorella kessleri in Escherichia coli.

To study the effect of sterols on the activity of the eukaryotic plasma membrane transporter, the hexose-proton symporter HUP1 from the unicellular alga Chlorella kessleri was expressed in Escherichia coli, a prokaryotic microorganism containing virtually no sterols. Under certain conditions, the recombinant protein was partially active in this prokaryotic organism. The heterologously produced HUP1p was purified from membrane fractions of E. coli and reconstituted in an in vitro system. The presence of ergosterol during solubilization, purification and reconstitution resulted in an increased activity of the reconstituted protein. Its activity, however, was 5-6 times lower as compared to the activity of HUP1p produced in Saccharomyces cerevisiae membranes and solubilized, purified, and reconstituted under the same conditions as above.

Post-translational fate of CAN1 permease of Saccharomyces cerevisiae.

To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. it was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones. Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition.

On the unidirectionality of arginine uptake in the yeast Saccharomyces cerevisiae.

The reversibility of arginine accumulation was followed in exponentially growing cells of Saccharomyces cerevisiae and in the same cells transferred to non-growing energized conditions. Under non-growing conditions the accumulated arginine is retained in the cells while in exponentially growing cells the accumulated radioactivity is released after the addition of high external concentrations of arginine. There are indications that the process is saturable. The accumulated arginine is not exchanged for other related amino acids (L-citrulline, L-histidine). Only L-lysine (a low-affinity substrate of the specific arginine permease) provokes partial radioactivity efflux from the cells. The switch of the arginine-related radioactive label efflux to its complete retention in the cells after changing the growth conditions occurs within a few minutes and is tentatively attributed to two concomitantly occurring events: (1) the actual presence of radioactive arginine (not its metabolite(s)) in the cell and (2) a modification of the specific arginine permease. The specific exchange of arginine described in the present study contrasts with the currently widely accepted opinion of unidirectionality of amino acid fluxes in yeast. The reasons why this phenomenon has not been observed before are discussed.

Possible nystatin-protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A Förster energy transfer study.

The Förster energy transfer from tryptophan residues of membrane proteins to nystatin was measured in reconstituted yeast plasma membrane vesicles free of, or doped with, ergosterol. We wanted to elucidate whether the functional change of membrane transport proteins from H+ symporters to facilitators, observed in ergosterol-containing plasma membrane vesicles on addition of nystatin [Opekarová and Tanner (1994) FEBS Lett. 350, 46-50], is reflected in altered protein-nystatin relations within the membrane. Both frequency-domain and time-domain time-resolved fluorescence spectroscopy showed that in the presence of ergosterol nystatin is located much closer to membrane proteins than in its absence.

Hexose/H+ symporters in lower and higher plants.

A well-studied transporter of plant cells is the hexose/H+ symporter of the unicellular alga Chlorella kessleri. Its properties, studied in vivo, are briefly summarized. In part, they are atypical and it has been suggested that this porter acts in an asymmetric way. Three genes coding for Chlorella hexose transport activity have been identified (HUP1, HUP2 and HUP3). HUP1 cDNA expressed in a mutant of Schizosaccharomyces pombe not transporting any D-glucose has been studied in detail. Several mutants with changed Km values for substrate were obtained, some by random polymerase chain reaction mutation and selection for decreased sensitivity towards the toxic sugar 2-deoxyglucose, some by site-directed mutagenesis. The amino acids affected clustered in the centre of the putative transmembrane helices V, VII and XI. Large families of hexose transporter genes are found in higher plants (Arabidopsis, Chenopodium, Ricinus). Their functional role is discussed. Finally, the progress made in studying plant transporters in a vesicle system energized by cytochrome c oxidase is summarized.

Nystatin changes the properties of transporters for arginine and sugars. An in vitro study.

In ergosterol-containing energized yeast plasma membrane vesicles nystatin (5-10 micrograms/mg total lipid) caused a massive efflux of pre-accumulated arginine while the membrane potential (the principal driving force; -110 mV) decreased by only 10-30 mV. Neither the substrate fluxes nor the membrane potential was influenced by nystatin when the permease was reconstituted in ergosterol-free phospholipid vesicles. The same effect of nystatin was found with the reconstituted sugar transporter from Chlorella kessleri. It is suggested that nystatin binding to ergosterol in the vicinity of the permease releases the transport protein from its coupling to energy and converts it to a facilitator.

The HUP1 gene product of Chlorella kessleri: H+/glucose symport studied in vitro.

An in vitro system was established to measure secondary active transport mediated by plant H+ symporters. For this purpose plasma membranes of Schizosaccharomyces pombe cells transformed with the HUP1 gene coding for the H+/hexose symporter of Chlorella kessleri were fused with cytochrome-c oxidase containing proteoliposomes. After energization with ascorbate/TMPD/cytochrome c these vesicles built up a protonmotive force of > 130 mV consisting mainly of a membrane potential of > 100 mV (inside negative). Energized vesicles accumulated D-glucose in a pH-dependent way up to 30-fold which was not the case with control vesicles prepared from cells transformed with the plasmid not containing the HUP1 gene. The Km value for D-glucose uptake was 5 x 10(-5) M. The pH-dependence of accumulation was not due to a difference in protonmotive force, but reflected the pH-dependence of the carrier activity, i.e., the accumulation was determined by kinetic and by thermodynamic parameters. In the system both components of protonmotive force delta psi and delta pH can be manipulated individually, which allows to evaluate to what extent they contribute to sugar accumulation. The results indicate that under certain conditions the internal pH may be a limiting factor for D-glucose accumulation.

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-beta-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 Ni(2+)-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.

Unidirectional arginine transport in reconstituted plasma-membrane vesicles from yeast overexpressing CAN1.

Amino acids are accumulated in Saccharomyces cerevisiae by strictly unidirectional influx systems. To see whether cellular compartmentation causes this unusual amino-acid-transport behaviour, arginine transport was studied in plasma-membrane vesicles. The arginine permease gene CAN1 was overexpressed in S. cerevisiae RH218a and in a permease-deficient mutant RS453 (can1). Reconstituted plasma-membrane vesicles from these transformants, energized by incorporated cytochrome-c oxidase, showed 3-4-fold increased rates of arginine uptake compared to vesicles from wild-type cells. The KT values were 32.5 microM in vesicles from wild-type and 28.6 microM in vesicles from transformed cells; the corresponding in vivo values were 17.5 microM and 11.4 microM, respectively. It could be demonstrated that unidirectional arginine transport and accumulation also exist in vesicles; thus, unidirectional influx is not related to cellular compartmentation.

Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae.

Yeast plasma membrane vesicles were obtained by the fusion of liposomes with purified yeast membranes by means of the freeze thaw-sonication technique. Beef heart mitochondria cytochrome-c oxidase was incorporated into the vesicles. Addition of substrate (ascorbate/TMPD/cytochrome c) generated a membrane potential negative inside, and an alkaline pH gradient inside the vesicle, that served as the driving force for leucine transport. Both delta pH and delta psi could drive leucine transport. When delta pH was increased in the presence of valinomycin and potassium, at the expense of delta psi, leucine uptake increased by 10%.

Long-term preservation of active luminous bacteria by lyophilization.

A simple method for long-term preservation of luminous bacteria is described. Cells of Vibrio fischeri, Photobacterium leiognathi and four strains of P. phosphoreum were suspended in a protective medium of low ionic strength (1% NaCl) supplemented with 15% lactose and 2% soluble starch, and lyophilized. The freeze-dried preparations were sealed under vacuum and stored at 4 degrees C. Luminous bacteria were resuscitated after six months by adding 2% NaCl up to the original volume. The rehydrated cells exhibited 16-28% of initial bioluminescence so that they could be used for a microbial test of toxicity (the Microtox test). This method is also useful for maintaining luminous bacteria in strain collections.