13] Thiamin transporters in yeast

Abstract

This chapter examines the thiamin transporters in yeast. The initial rate of [ 14 C]thiamin uptake by Saccharomyces cerevisiae ( S. cerevisiae )grown at exponential phase in thiamin-free Wickerham's synthetic medium is approximately 10-fold higher than that by commercially available bakers' yeast. The uptake by the former cells is linear for about 2 min, whereas that by the latter cells is for about 20 min. Thiamin accumulation is mediated by a single active transport system operating with a pH optimum of 4.5–5.0 and an apparent K m value for thiamin of 0.18 μ M. Two thiamin-binding proteins have been found in S. cerevisiae —one is a soluble thiamin-binding protein (sTBP) in the periplasmic space, and the other is bound to plasma membrane (mTBP). The thiamin-binding activities of these proteins can be assessed by a standard equilibrium dialysis method. The so-called constitutive acid phosphatase localized in the yeast periplasmic space is repressed by exogenous thiamin. Because both thiamin transport and the activity of sTBP are repressed by pregrowth of yeast cells in thiamin-containing medium, the activity of the thiamin-repressible acid phosphatase is also measured throughout the purification of sTBP.