Deletion strains lacking the TPS1 gene encoding Tps1p, an essential component of the trehalose synthase complex, cannot synthesize any trehalose (Bell et al., 1998). They are unable to acquire thermotolerance after preconditioning at 37°C followed by thermal insult at 48–50°C, and are thus unable to form colonies when plated at physiological temperature 24°C, whereas most normal cells survive such thermal treatments (see Piper, 1998). In Δtps1 cells, a β-lactamase fusion protein residing in the ER lumen was inactivated by exposure of preconditioned cells briefly to 48–50°C. After shift of the cells back to physiological temperature, β-lactamase was not refolded to an enzymatically active and secretion-competent form, like in normal cells, but remained in the ER in an inactive form. Refolding of a heat-affected natural glycoprotein, the vacuolar protease pro-CPY to a transport-competent form in the ER was severely delayed in the absence of trehalose synthesis. The Δtps1 mutants remained viable for hours after severe heat stress and endocytozed the plasma membrane with similar kinetics as WT cells, although electron microscopy revealed aggregates in the cytosol and protein synthesis was blocked. Thus, trehalose appears to facilitate conformational repair of heat-damaged glycoproteins in the ER lumen. We have demonstrated a similar function for the cytosolic chaperone Hsp104 (Hänninen et al., 1999). Is it possible that trehalose and Hsp104 visit the ER lumen to assist conformational repair events? Trehalose synthase is a cytosolic enzyme complex and both trehalose and Hsp104 have been detected exclusively in the cytosol and the nucleus (Singer and Lindquist, 1998b). Hsp104 is a homohexameric complex (Schirmer et al., 1996), too large to pass the ER translocon, which has been estimated to have a maximal diameter of 60 Å (Hamman et al., 1997). Hsp104 has several potential N-glycosylation sites, whose glycosylation might reveal passage of monomeric unfolded molecules into the ER, however, no glycosylated variants have been detected. Passage of trehalose into the ER lumen would require a transporter. The yeast genome sequence does not support the existence of such a transporter. Thus, the effects of Hsp104 and trehalose must be exerted across the ER membrane. Perhaps a transmembrane protein of the ER membrane, required for intraluminal repair functions, is heat-damaged, and Hsp104, together with trehalose, repairs this domain to restore the ER refolding function. Hsp104 with functional ATP-binding sites and trehalose are required for refolding events in the cytosol (Singer and Lindquist, 1998a), as well as in the ER lumen (Hänninen et al., 1999). Our data do not rule out more specific cross-talk between the cytosolic and luminal chaperone machineries.
Heat has been proposed to have multiple effects on biological membranes (see Piper, 1997), and trehalose has been proposed to protect membranes from environmental stress conditions (Crowe et al., 1984, 1992). Thus, we examined membrane traffic functions in heat-treated cells in the presence and absence of trehalose synthesis in vivo. The rate of endocytosis and exocytosis of the plasma membrane uracil permease was similar before and after severe heat stress, as well as in the presence and absence of trehalose synthesis. A lipophilic membrane marker, FM 4-64, revealed a heat-sensitive step along the endocytotic pathway. Soon after severe heat stress, the plasma membrane was internalized, but delivery of the membrane to the vacuole was delayed. However, this was a feature of both Δtps1 and WT cells. We conclude that trehalose had no role in protection against heat-inflicted damage of membranes engaged in vesicular traffic in vivo under our experimental conditions.