l-Proline transport in Saccharomyces cerevisiae

Abstract

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s⁻¹ · (g dry wt.)⁻¹, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s⁻¹ · (g dry wt.)⁻¹, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.