Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Abstract

Summary. The specific binding activity to [laC]thiamine was found to be located in the plasma membrane of Saccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant of Saccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.

Photoaffinity labeling of thiamin‐binding component in yeast plasma membrane with [3H]4‐azido‐2‐nitrobenzoylthiamin

“…The optimal pH of the binding reaction was 5.0. This value is compatible with the optimal pH of thiamin uptake by yeast cells [2] and thiamin binding by the previously prepared membrane fraction [5]. The binding activity was decreased more than 50070 at a pH value below 4 or above 6 and was lost completely at pH 7.…”

“…Fig. 1 shows that the binding activity was increased linearly with increasing thiamin concentration [5]. Acquisition of the plasma membrane fraction including many thiamin-binding components seems to result from the preparation of yeast protoplasts, followed by homogenization of the protoplasts with glass beads in a beadbeater.…”

“…Recently, we have reported the occurence of a membrane-bound thiamin-binding protein in S. cerevisiae, besides a soluble thiamin-binding protein in the periplasm [3,4]. Furthermore, it has been disclosed that the thiamin-binding protein located in the plasma membranes of S. cerevisiae plays an important role in the thiamin uptake and some properties of thiamin binding by the plasma membranes have also been described [5]. However, the protein components playing the uptake role in the cell membranes have remained unclear until the present time.…”

Isolation and Characterization of a Thiamin Transport Gene,THI10, from Saccharomyces cerevisiae

“…Thiamin Binding Activity of THI10 Protein-We have previously reported the occurrence of a thiamin binding activity in the plasma membrane of S. cerevisiae (20,28). The thiamin binding activity is repressed by exogenous thiamin, and the dissociation constant (K d ) value for thiaminis of 0.11 M is close to the apparent K m (0.18 M) of thiamin transport in S. cerevisiae, but the entity of the protein in the cell membranes remains unclear.…”

“…The thiamin binding activity was determined by an equilibrium dialysis against 10 Ϫ7 M [ 14 C]thiamin overnight in the cold as described (19) after subtracting nonspecific thiamin binding performed in the same manner except that 10 Ϫ4 M nonlabeled thiamin was added in the outer solution of dialysis containing the labeled thiamin. Yeast plasma membrane was prepared by a sucrose gradient centrifugation method from the crude membrane fraction as previously reported (20). Protein concentration in membrane preparations was measured by the procedure of Markwell et al (21) using bovine serum albumin as standard.…”