Jean Chevallier

Characterization of the Saccharomyces cerevisiae cytosine transporter using energizable plasma membrane vesicles.

The purine-cytosine permease is a carrier localized in the plasma membrane of the yeast Saccharomyces cerevisiae. The energetics of cytosine transport catalyzed by this permease has been studied in an artificial system obtained by fusion between proteoliposomes containing beef heart cytochromec oxidase and plasma membrane-enriched fractions of aS. cerevisiae strain overexpressing the permease. Upon addition of an energy donor, a proton-motive force (inside alkaline and negative) is created in this system and promotes cytosine accumulation. By using different phospholipids, it is shown that cytosine uptake is dependent on the phospholipids surrounding the carrier. It was demonstrated that the purine-cytosine permease is able to catalyze a secondary active transport of cytosine. By using nigericin and valinomycin, the ΔpH component of the proton-motive force is shown to be the only force driving nucleobase accumulation. Moreover, transport measurements done at two pH values have shown that alkalinization of intravesicular pH leads to a significant increase in cytosine uptake rate. Finally, no specific role of K+ ions on cytosine transport could be demonstrated in this system.

Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles

A study of electrokinetic properties of reconstituted sarcoplasmic reticulum was undertaken to determine the nature of the groups bearing the negative charge of the membrane. After incorporation of phosphatidylcholine into the bilayer, it was found that the Ca2+-ATPase embedded in functional vesicles bore 3e- per mole. When the surface charge density of the hydrodynamic particles became more negatively charged by incorporation of phosphatidylserine molecules, the reconstituted vesicles had a tendency to build large structures resulting from vesicle-vesicle interaction and containing large amounts of divalent cations. These aggregated structures may partially explain the discrepancy observed between the expected value of the surface charge density and the data obtained by electrophoretic mobility measurements. This work emphasizes the importance of a renewal of the classical interpretation of electrophoretic mobility data in order to analyze the results obtained with biological material. To explain the energy transduction process which takes place in the sarcoplasmic reticulum membrane, it was of interest to determine whether or not variations of the surface electrical properties affect the calcium ion translocation upon ATP hydrolysis. Relatively significant modifications of the bilayer composition and surface charge density did not appreciably affect the calcium transport activity.

Simultaneous preparation of membrane fractions from small amounts oe skeletal muscle: A study on mitochondrial and microsomal fractions from Med mice

This work is the first biochemical study of skeletal muscle membranes isolated from mice displaying an inherited neuromuscular disease: MedJ strains. It is focused on the research of a possible alteration of membrane biological activities related to this disease. We describe a procedure which allows the simultaneous preparation of mitochondrial and microsomal fractions from a small amount of skeletal muscle. When EGTA and BSA are present in the buffers, functional mitochondria can be prepared. Under these conditions we found that no major modification occurs for this disease at the mitochondrial inner membrane level. A dramatic impairment of a calcium active transport activity found in the microsomal fraction obtained from MedJ is noticed, suggesting that some modification may occur at this level.

Functional analyses of yeast purine-cytosine permease mutants

The purine-cytosine permease (PCP) is an active transporter located in the plasma membrane o f the yeast Saccharomyces cerevisiae. This protein mediates purines (adenine, guanine and hypoxanthine) and cytosine accumulation in the cell using an electrochemical potential gradient o f protons as energy source. In order to study the structure-function relationship of PCP, various mutant strains, showing altered transport o f one or several bases, were selected [1]. Their cloning and sequencing revealed that three o f them presented substitutions in the same region o f the putative sequence o f the PCP; this region would correspond to the hydrophil ic segment I371 -A-N-NI -P -N377[2]. Surprisingly, one o f these spontaneous mutants ( thefcy2-21 allele) displayed three amino acid changes (I371V , I375V and N377G ). Therefore, in order to analyze the contribution o f individual amino acid changes to the phenotype o f the mutant fcy2-21, single (N377G) and double (I371V , I375V ) mutants were constructed by si te-directed mutagenesis [2]. For further biochemical studies, we cloned all o f these mutated alleles into a muir• plasmid called pJDB207 [3]. The corresponding plasmids were introduced in a Saccharomyces cerevisiae strain carrying a chromosomal deletion at the FCY2 locus (NC233-10B), as already done for the wild type allele [4]. Determination o f the uptake constants o f purine bases and cytosine, i.e. the apparent Michaelis constant o f transport (Kf) and the maximal rate of uptake (Vm), showed that the phenotype o f the triple mutant is almost the same as the NC233-10B; pTF2 one, which carries a single substitution N377G (data not shown). This mutation leads to a dramatic increase o f the /~ t for cytosine and hypoxanthine as compared to the wild type strain (NC233-10B; pAB4). On the other hand, the phenotype o f the double mutant NC23310B; pTF1 strain, carrying the two substitutions I371V and I375V, is comparable with the wild type one. These results are quite similar to those already obtained with non-amplified strains [2].

The use of auto-anti-idiotypes for the visualization of acetylcholine receptors in an invertebrate skeletal muscle

Polyclonal auto-anti-idiotypic antibodies (AAIs), detected in antisera of rabbits immunized with acetylcholine conjugate, have already been characterized. In this paper, we report on a cytochemical application of these AAIs on skeletal muscle motor endplates of an invertebrate. We have shown that there is a non-uniformly distributed reaction in the synaptic cleft. The deposits seem to be associated with the muscular plasma membrane. The specificity of the cytochemical method is discussed. On this kind of muscle, the AAIs are able to detect, at the ultrastructural level, acetylcholine binding sites which most likely are acetylcholine receptors.

Quantitative determination of the calcium involved in the regulation of the Ca2+-ATPase activity in sarcoplasmic reticulum vesicles.

The dependence of the Ca2+-ATPase activity of sarcoplasmic reticulum vesicles upon the intravesicular concentration of calcium accumulated after active uptake was studied. The internal calcium concentration was modified by addition of the ionophore A23187 at the steady state of accumulation. About half of the calcium accumulated could be released at low ionophore concentration without any concomitant activation of the Ca2+-ATPase. This population of calcium might consist of calcium free in the lumen of the vesicles or bound to the bilayer at sites which do not interact with the ATPase activity. At higher concentrations of ionophore (above 1.75 nmol A23187/mg protein) the release of calcium activated this enzyme. This phenomenon was independent of the extravesicular calcium concentration and might be explained by assuming second species of calcium ions bound to the inner side of the membrane and in close functional interaction with the Ca2+-ATPase.