Christian Napias

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.

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].