Amino acid uptake by yeasts: IV. Effect of thiol reagents on l-leucine transport in Saccharomyces cerevisiae

(1) $\text{N-}\text{Ethylmaleimide}$ (a penetrating SH- reagent) inactivated l-[¹⁴C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with $\text{N-}\text{ethylmaleimide}$, $\text{N-}\text{ethylmaleimide}$ concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With d-glucose-energized yeast, $\text{N-}\text{ethylmaleimide}$ inhibited l-[¹⁴C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit l-[¹⁴C]leucine binding, except when the cells were preincubated with l-leucine. With the rho^? respiratory-deficient mutant (energized cells), $\text{N-}\text{ethylmaleimide}$ inhibited l[¹⁴C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the $\text{N-}\text{ethylmaleimide}$ effect as a function of l-[¹⁴C]leucine concentration showed a significant decrease of $\text{J}{}_{\mathrm{<mtext>max</mtext>}}$ values of the high- (S₁) and low- (S₂) affinity amino acid transport systems, but $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ values were not significantly modified. (3) When assayed in the presence of d-glucose, $\text{N-}\text{ethylmaleimide}$ inhibition of d-glucose uptake and respiration contributed significantly to inactivation of l-[¹⁴C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of l-[¹⁴C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate l-[¹⁴C]leucine entrance, while $\text{p-}\text{chloromercuribenzoate}$, a slowly penetrating SH- reagent, inactivated it to a limited extent. When compared with the effect of $\text{N-}\text{ethylmaleimide}$, these negative results indicate that thiol groups of the l-[¹⁴C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.