Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H + -ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 8C in the presence of 1 m trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t 0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 m trehalose, the t 0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K 0.5 ) decreased from 0.5 m, in the solubilized state, to 0.04 m in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H + -ATPase cleavage by trypsin changed when 1 m trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E 2 -like form. At elevated temperatures, cooperative intramolecular motions occur until a temperature is reached where noncovalent forces that maintain the native structure of the protein can no longer prevail against the increase in entropy [1±5]. As a result, the protein loses most of its ordered secondary and tertiary structures, and the protein is denatured. When a protein unfolds, hydrophobic regions that were located in the interior become exposed to the solvent, a situation that is thermodynamically unfavorable [1]. The thermal stability of a protein can be changed intrinsically by the alteration of amino acids or extrinsically by the addition of suitable stabilizing effectors, e.g. peptides, coenzymes, membranes and osmolytes [6±9].Organisms and cellular systems are required to adapt to stress conditions such as high temperature, often responding by accumulating organic solutes such as sugars, polyols, amino acids or methylamines [5,10±15]. This accumulation is associated with the effectiveness of these osmolytes in minimizing protein denaturation and membrane damage under stress conditions [16±21]. The ability of carbohydrates and methylamines to stabilize proteins has been attributed to the preferential hydration of proteins in carbohydrate solutions [7,22]. This phenomenon is due to the fact that polyols, in general, do not interact with the protein molecule [7,22] and are therefore called nondisturbing osmo...