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Saccharomyces cerevisiae cells grown at physiological temperature 24°C require preconditioning at 37°C to acquire tolerance towards brief exposure to 48–50°C. During preconditioning, the cytosolic trehalose content increases remarkably and in the absence of trehalose synthesis yeast cannot acquire thermotolerance. It has been speculated that trehalose protects proteins and membranes under environmental stress conditions, but recently it was shown to assist the Hsp104 chaperone in refolding of heat-damaged proteins in the yeast cytosol. We have demonstrated that heat-denatured proteins residing in the endoplasmic reticulum (ER) also can be refolded once the cells are returned to physiological temperature. Unexpectedly, not only ER chaperones but also the cytosolic Hsp104 chaperone is required for conformational repair events in the ER lumen. Here we show that trehalose facilitates refolding of glycoproteins in the ER after severe heat stress. In the absence of Tps1p, a subunit of trehalose synthase, refolding of heat-damaged glycoproteins to bioactive and secretion-competent forms failed or was retarded. In contrast, membrane traffic operated many hours after severe heat stress even in the absence of the TPS1 gene, demonstrating that trehalose had no role in thermoprotection of membranes engaged in vesicular traffic. However, cytosolic proteins were aggregated and protein synthesis abolished, resulting finally in cell death.
All organisms acquire tolerance towards otherwise lethal high temperatures if they are first preconditioned at a moderately high temperature where their heat shock genes are activated (Parsell and Lindquist, 1993). In Saccharomyces cerevisiae, Hsp104 is indispensable for acquisition of thermotolerance (Sanchez and Lindquist, 1990; Sanchez et al., 1992; Schirmer et al., 1996). Hsp104 is an HSP100/Clp ATPase with two ATP-binding sites, both of them essential for thermotolerance (Parsell et al., 1991). It promotes survival of yeast cells exposed to 48–50°C after preconditioning at 37°C by directly disaggregating heat-denatured proteins in the cytosol (Parsell et al., 1994).
The concentration of the non-reducing disaccharide trehalose increases remarkably in cells exposed to high temperatures, and trehalose is required for acquisition of thermotolerance in exponentially growing S. cerevisiae and Schizosaccharomyces pombe cells (Wiemken, 1990; de Virgilio et al., 1994; Ribeiro et al., 1997). Trehalose is synthesized by trehalose-6-phosphate synthase (Tps1p) and trehalose-6-phosphate phosphatase, which make part of a cytosolic enzyme complex (Bell et al., 1998). The primary lethal lesions caused by heat have not been identified, but it has been proposed that high temperature disrupts membranes and denatures proteins. Trehalose has been suggested to stabilize both proteins and membranes (Crowe et al., 1984, 1992). However, recently it was demonstrated that trehalose does not protect proteins from heat damage, but rather suppresses heat aggregation of proteins, maintaining them in a non-native state. Upon dilution from trehalose, the proteins are refolded by Hsp104 (Glover and Lindquist, 1998; Singer and Lindquist, 1998a). Trehalose is rapidly degraded after the heat stress is over, which is thought to promote the refolding function of Hsp104 (Singer and Lindquist, 1998b).
We have identified a novel function of the yeast endoplasmic reticulum (ER), namely conformational repair of in vivo heat-damaged proteins. The repair machinery involves the ER-resident Lhs1 protein (Jämsäet al., 1995; Saris et al., 1997), a member of the Hsp110 subgroup of the Hsp70s sharing 24% identical amino acids with the ER chaperone BiP/Kar2p (Rasmussen, 1994; Craven et al., 1996, 1997). In the absence of Lhs1p, previously heat-denatured proteins were not refolded, and in normal cells Lhs1p was found in association with heat-damaged proteins but not native copies (Saris et al., 1997; Saris and Makarow, 1998). Unexpectedly, also the cytosolic Hsp104 chaperone was found to be required for refolding of heat-denatured artificial and natural yeast proteins in the lumen of the ER (Hänninen et al., 1999). Here we show that trehalose, together with Hsp104, facilitates conformational repair of heat-damaged glycoproteins in the ER lumen. Here we also tested in vivo whether trehalose protects membranes from heat-inflicted damage. Endo- or exocytotic membrane traffic functioned similarly in heat-treated TPS1 and Δtps1 cells, demonstrating that trehalose had no role in thermoprotection of membranes involved in vesicular traffic in living yeast cells.
Heat-denatured Hsp150Δ-β-lactamase failed to be reactivated in the ER in the absence of trehalose synthesis
First, for reference, we show the reactivation of heat-inactivated Hsp150Δ-β-lactamase in cells which are normal for trehalose synthesis. The reporter protein, Hsp150Δ-β-lactamase, was placed under the control of a promoter which is upregulated when cells are shifted from 24°C to 37°C (Russo et al., 1993), and expressed in a sec18-1 mutant (strain H393; see Table 1 for genotypes and Table 2 for relevant features of strains used in terminal experiments). The Hsp150 fragment is a signal peptide-containing, 321-amino-acid long N-terminal fragment of the natural secretory yeast glycoprotein Hsp150 (Russo et al., 1992). It helps the β-lactamase portion to fold properly in the yeast ER (Simonen et al., 1994), and provides O-glycosylation sites for distinction of the biosynthetic intermediates (Paunola et al., 1998). In the sec18-1 mutant, membrane traffic from the ER to the Golgi is normal at 24°C, but reversibly inhibited at 37°C (Novick et al., 1981). Thus, when sec18-1 cells grown at 24°C were incubated for an hour at 37°C (Fig. 1B), the synthesis of the reporter was upregulated, the newly synthesized and translocated, catalytically active reporter molecules accumulated in the ER due to the sec18-1 mutation, and the cells acquired thermotolerance towards brief exposure to 48–50°C. A subsequent 20 min incubation at 50°C inactivated the enzyme (Fig. 1B; see panel A for thermal treatments). However, when the cells were returned to 24°C and cycloheximide (CHX) was added to stop protein synthesis, more than 70% of the original β-lactamase activity was resumed in about 4 h (closed circles).
To study whether trehalose had any role in the reactivation of heat-denatured β-lactamase, the above experiment was repeated using a mutant where the TPS1 gene was deleted (strain H881, Δtps1 sec18-1), resulting in complete lack of Tps1 activity and trehalose synthesis (Bell et al., 1998). In the Δtps1 sec18-1 mutant, the 37°C incubation resulted in intracellular accumulation of β-lactamase activity, and the 50°C treatment inactivated the enzyme, as above. However, a subsequent recovery period at 24°C in the presence of CHX resulted in only very modest reactivation of the reporter (Fig. 1C, closed circles). Strain H941, in which the chromosomal HSP104 gene was replaced by a variant carrying a point mutation that destroys one of its two ATP-binding sites, (hsp104K218Tsec18-1) served as a control: refolding of heat-denatured Hsp150Δ-β-lactamase occurred to only a very low degree (Fig. 1D). In all cases, addition of the metabolic inhibitor NaN3 to the recovery mixture inhibited reactivation (Fig. 1B–D, broken lines). In none of the strains was β-lactamase activity secreted to the medium during the entire experiment (not shown).
It was essential to verify that in the Δtps1 sec18-1 mutant the reporter protein had been properly translocated and resided in the ER at the time of the thermal insult at 50°C. After preincubation for 15 min at 37°C, which closes the sec18 block, sec18-1(Fig. 2A), Δtps1 sec18-1 (Fig. 2B) and hsp104K218T,K620Tsec18-1 (Fig. 2C) cells were pulse labelled with [35S]-methionine/cysteine for 5 min at 37°C (lanes 1). Immunoprecipitation with β-lactamase antiserum and SDS–PAGE analysis showed that in each case part of the protein was in the ER, because it migrated as a form with primary O-glycans (110 kDa). Slightly more than half migrated as the unglycosylated 66 kDa form and was thus still in the cytosol. After a 5 min chase at 37°C, more protein was in the ER form (lanes 2), and after a chase of 20 min, almost all resided in the ER in all three strains (lanes 3). Thus, the reporter was translocated into the ER with similar kinetics in the absence and presence of Tps1p. We have demonstrated earlier that the Hsp150 signal peptide confers very slow post-translational translocation, and verified the subcellular localization of the various Hsp150Δ-β-lactamase intermediates (Paunola et al., 1998). Moreover, indirect immunofluorescent staining with β-lactamase antiserum revealed the reporter molecules in ER-like structures in sec18-1(Fig. 3A), Δtps1 sec18-1 (Fig. 3B) and hsp104K218T,K620Tsec18-1 (Fig. 3C) cells after preconditioning at 37°C. The Δtps1 strain was vulnerable towards fixatives, resulting in a punctate-like staining. Immunostaining of hsp104K218T,K620Tsec18-1 cells with Lhs1p (ER resident protein) antiserum, highlighted the ER which appears in 37°C-treated sec18-1 cells as a rim beneath the plasma membrane (Fig. 3D). We have earlier shown that heat-denatured Hsp150Δ-β-lactamase persisted in the ER form in the absence of Hsp104 function (Saris et al., 1997). This was also the case in the absence of Tps1 function (not shown).
Refolding of pro-CPY to a secretion-competent conformation required trehalose synthesis
To confirm the physiological importance of trehalose in refolding events in the ER, we extended our studies from the artificial β-lactamase reporter to an authentic yeast glycoprotein carboxypeptidase Y (CPY). In the ER, pro-CPY acquires four primary N-glycans (see Fig. 4, p1, 67 kDa), which are extended in the Golgi (p2, 69 kDa) in normal cells. After transport to the vacuole, the pro-sequence is removed yielding mature CPY of 62 kDa (Stevens et al., 1982). A Δtps1 deletion strain was preincubated at 37°C for an hour and then 35S-labelled for 5 min (Fig. 4A, lane 1). Immunoprecipitation and SDS–PAGE analysis showed that the protein resided in the ER because it occurred in the pro-form. In these experiments, normal cells lacking sec mutations were used. Because pro-CPY translocates rapidly and leaves the ER slowly, it is mostly in the ER after a 5 min pulse, and thus ER-blocking mutations could be omitted. Parallel similarly labelled samples were thereafter incubated for 20 min at 48°C (lane 2) and chased then at 24°C as indicated. Phosphoimager quantification showed that half of the molecules were in the vacuole after about 3 h 20 min (Fig. 4E, closed circles). In normal cells, pro-CPY was transported to the vacuole much faster, as half of the molecules were there in roughly 1 h 20 min (Fig. 4B and E, open circles). In an hsp104 mutant lacking functional ATPase sites (hsp104K218T,K620T), pro-CPY remained in the ER (Fig. 4C). Thus, in the absence of Tps1p, heat-affected pro-CPY acquired a transport competent structure much more slowly than in cells with normal trehalose synthesis. Because Δtps1 mutants cannot grow on glucose, in all Δtps1 strains we used the HXK2 gene was also deleted, thus rescuing the ability to grow in glucose-containing media (indicated where relevant, e.g. in Table 2; Blàzquez and Gancedo, 1994). Although HXK2 has not been found to have a role in acquisition of thermotolerance (see below), we repeated the above experiment on strain H838 (Δhxk2). The kinetics of resumption of secretion competence of heat-damaged pro-CPY (Fig. 4D) were similar as in wild-type (WT) cells (Fig. 4B).
One explanation for retarded refolding of pro-CPY is that in the absence of trehalose the cells lost rapidly viability. Or, the exocytic membranes were damaged in the absence of trehalose and slow maturation of pro-CPY was caused by extremely slow membrane traffic. These alternatives are addressed below.
Metabolic activity and protein synthesis in Δtps1 mutants after severe heat stress
In the absence of trehalose synthesis, S. cerevisiae cells cannot form colonies when preconditioned at 37°C and are subjected briefly to 48–50°C. Thus, the failure of reactivation of heat-denatured β-lactamase reporter in Δtps1 mutants (Fig. 1C), could be explained by abrupt cell death after thermal insult. First, we confirmed directly that Δtps1 cells, which had undergone preconditioning at 37°C and a 48°C treatment (Fig. 5A), were metabolically active: they consumed glucose (Fig. 5B, open circles) like normal cells (closed circles) for at least 6 h after severe heat stress. However, bulk protein synthesis, measured as incorporation of [35S]-methionine/cysteine into TCA-precipitable material, was inhibited apparently irreversibly by the thermal insult in Δtps1 cells (Fig. 6B, dark columns). Because these cells lacked, in addition to the TPS1 gene, also the HXK2 gene, we used Δhxk2 cells as a control in both experiments. In Δhxk2 cells, protein synthesis started slowly after the 50°C treatment and was normal after 4 h (whole columns). Lack of protein synthesis per se, however, did not prevent the renaturation of heat-damaged β-lactamase reporter in TPS1 cells (see Fig. 1B). Finally, we confirmed that deletion of the HXK2 gene alone had no effect on acquisition of thermotolerance. Indeed, after preconditioning at 37°C and exposure to 50°C (see Experimental procedures), 26% of Δhxk2 cells (H838) and 30% of WT cells (H1) formed colonies, whereas less than 1% of the Δtps1Δhxk2 mutant (H837) survived the heat treatments.
Endocytosis in heat-treated cells in the absence of trehalose synthesis
We needed to confirm that the slow transport of heat-affected pro-CPY to the vacuole during the recovery period in Δtps1 mutant (Fig. 4A) was not due to retarded membrane traffic. Exocytosis after severe heat stress could not be studied in the Δtps1 mutant by following intracellular transport of reporter proteins synthesized during the recovery period because of the cessation of protein synthesis. Thus, we studied internalization of the plasma membrane as a measure of membrane traffic. Uptake of uracil from the medium depends on the availability of uracil permease in the plasma membrane. Normally, the permease is constantly internalized and degraded in the vacuole, while new molecules are synthesized and transported to the plasma membrane via exocytosis. Inhibition of incorporation of new molecules by CHX allows the determination of the rate of endocytosis of uracil permease by measuring the uptake of [14C]-uracil (Volland et al., 1992). In Δtps1 and WT cells, 50% of the permease was internalized, in the presence of CHX, in about 65 min in untreated control cells at 24°C (Fig. 7A and B, closed circles). Parallel cells were preconditioned at 37°C, treated for 15 min at 48°C, and then assayed for uracil uptake at 24°C. Uracil permease was internalized with almost similar kinetics as in untreated cells (open circles). Also in Δhsp104 cells (Fig. 7C), uracil permease was internalized similarly before and after the thermal treatments, although somewhat more slowly than in Δtps1 and WT cells. When ATP was depleted with sodium azide, [14C]-uracil uptake was completely inhibited (Fig. 7B, squares). Thus, internalization appeared to operate after severe heat stress whether or not the cells were able to synthesize trehalose. Because CHX was present during the experiment, the components driving internalization appeared not to be damaged by the thermal insult, or if some were, they were rapidly repaired by pre-existing chaperones. As described in Fig. 7 legend, roughly half of the permease molecules could be detected at the cell surface immediately after shift of the cells from 48°C to 24°C (zero time points, open circles), as compared with the untreated control (zero time points, closed circles). This was due to the fact that the rate of endocytosis of the permease is accelerated at 37°C (Volland et al., 1994), and thus the preconditioning period at 37°C preceding the thermal insult at 48°C depleted half of the molecules from the plasma membrane.
Whether the permease molecules reached only the endosomes, or were transported all the way to the vacuole, was not revealed by these data. To analyse whether also endosome-to-vacuole traffic operated in heat-treated cells, we used the lipofilic drug FM 4-64, which stains the plasma membrane and is then incorporated into the vacuolar membrane via endocytosis (Vida and Emr, 1995). First, we incubated WT cells with FM 4-64 in the cold to prevent internalization. Fluorescent microscopy of the live cells showed bright staining of the surface (Fig. 8A, fluorescent images on the left and difference interference contrast (DIC) on the right). When the cells were treated with FM 4-64 at 24°C to allow internalization, the vacuolar membrane was stained (Fig. 8B). Evidently, the stained plasma membrane had been endocytozed and the endosomes fused with the vacuole. When the cells were preconditioned at 37°C, shifted then for 20 min to 48°C and treated thereafter with FM 4-64 at 24°C, the drug appeared to be internalized. However, it did not stain the vacuolar membrane but resided in the cytosol, presumably in prevacuolar compartments (Fig. 8C). When cells treated at 37°C and 48°C were allowed to recover for 3 h at 24°C before incubation with FM 4-64 at 24°C, the vacuole membrane was stained (Fig. 8D), indicating that endocytosis from the plasma membrane to the vacuole operated. In the Δtps1 mutant, similar results were obtained (Fig. 8E–G). We suggest that internalization functioned right after the thermal insult, but traffic from the prevacuolar compartment to the vacuoles was first inhibited or retarded, and resumed after some hours of recovery. Trehalose appeared to have no role in resumption of the endocytotic functions after severe heat stress.
Exocytosis after severe heat stress
In the endocytosis experiments, we used yeast strains that had no sec mutations. It turned out that exocytosis could also be studied when the uracil permease internalization assay was performed on cells harbouring the sec18-1 mutation. The Δtps1 sec18-1 mutant was preconditioned for an hour at 37°C, which resulted in ER accumulation of newly synthesized exocytic proteins, also uracil permease. The cells were then treated at 48°C, shifted back to 24°C, and the surface exposure of uracil permease was assayed as above (Fig. 9, open circles). First, the permease started to be internalized. However, after an hour the amount at the plasma membrane increased to almost double the amount detected at the beginning of the internalization assay. This must have been due to reversion of the transport block at 24°C, resulting in transport to the plasma membrane of the ER-accumulated pool of permease molecules. Thereafter, the amount of the permease at the surface decreased by 75% in 2 h, with similar kinetics as in untreated control cells (closed circles). Again, as in Fig. 7, about half of the permease molecules had been internalized during the preconditioning period at 37°C (zero time point, open circles). Similar data were obtained for sec18-1 cells which synthesized trehalose normally (H974), except that the increase of the permease at the surface was not as steep (not shown). These data demonstrate that exocytosis from the ER to the plasma membrane operated in Δtps1 cells at least after 1–2 h of recovery at 24°C, and that trehalose was not required for maintenance of exocytic membrane functions after thermal insult. The heat-treated Δtps1 mutant, however, could not synthesize anymore proteins (see above), which manifested itself as lack of colony formation.
Finally, transmission electron microscopy was used to examine the heat-treated cells. After growth at 24°C, no obvious differences in cellular morphology could be detected between Δtps1 cells (Fig. 10A) and cells harbouring the TPS1 gene (Fig. 10C). However, after preconditioning at 37°C, thermal insult for 20 min at 48°C and recovery for 6 h at 24°C, the cytosol of Δtps1 cells was full of aggregate-like structures (Fig. 10B, open arrows), which were absent from the control cells (Fig. 10D), whereas the nuclear, vacuolar and ER membranes of heat-treated Δtps1 and WT cells appeared similar.
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.
Strains and media
The following previously described yeast strains were used in this study (genotypes in Table 1): H1, H4, H393 (Simonen et al., 1994), H454 (Sanchez and Lindquist, 1990), H777 (Schlenstedt et al., 1995), H835 (Hänninen et al., 1999), H837, H838 (Blàzquez and Gancedo, 1994), H851 and H941(Hänninen et al., 1999). Strains H881 and H882 were created by mating strains H393 and H837 and dissecting the spores of the resulting diploid. Strains H982 and H984 were obtained by transformation of strains H454 and H851 with integrative plasmid pKTH4753 (see below) respectively. Strains H966, H974, H990, H992 and H993 were created by transforming the multicopy plasmid p195gF (2 µURA3 GAL-FUR4) (Volland et al., 1994) containing the FUR4 gene under the control of the GAL10 promoter into strains H1, H4, H454, H837 and H882 respectively. Transformations were carried out with the lithium acetate method (Hill et al., 1991). Yeast strains were grown at 24°C in shake flasks overnight to early logarithmic phase in YPD medium, in synthetic complete (SC) medium lacking methionine and cysteine, or in SC medium lacking uracil and containing 4% galactose and 0.02% glucose. E. coli strain DH5α (Sambrook et al., 1989), used as cloning host, was grown in Luria–Bertani (LB) medium supplemented with ampicillin (100 µg ml−1).
Site-directed mutagenesis of ATP-binding sites of Hsp104.
A mutant variant of HSP104 with point mutations in both ATP-binding sites was generated by site-directed mutagenesis. PCR was used to introduce mutation K620T to the Hsp104K218T mutant as described before for the latter mutation (Hänninen et al., 1999), using primers 82009 (5′-GGTTCCGGTACAACTGAATTGG-3′) and 82216 (5′-CCAATTCAGTTGTACCGGAACC-3′) as the mutagenizing primers and pKTH4719 (Hänninen et al., 1999) as the template. After the second PCR, the resulting fragment was digested with ClaI and SpeI (Promega) and inserted into pKTH4685 to produce plasmid pKTH4751. The plasmid pKTH4753 expressing Hsp104K218T,K620T was constructed by cloning the 3.8 kb SacI–SalI fragment from pKTH4751 to pFL34 (Bonneaud et al., 1991) as described for pKTH4720 (Hänninen et al., 1999), and integrated into hsp104 deletion strain H454 to create strain H982, and to the hsp104 deletion strain H851 expressing Hsp150Δ-β-lactamase to create H984.
Uracil permease endocytosis assay
The uracil permease internalization assay was carried out according to Volland et al. (1992). The FUR4 overexpression strains containing the episomal multicopy plasmid p195gF (2 µURA3 GAL-FUR4) (Volland et al., 1994) were grown in SC medium lacking uracil and containing 4% galactose and 0.02% glucose to OD600 0.2–0.4. CHX was added to the final concentration of 100 µg ml−1, and duplicate 1 ml samples were incubated with 5 μM uracil containing 25 nM [14C]-uracil (NEN Life Science Products) for 2 min at 24°C and quickly passed through a Whatman GF/C filter. The filter was washed with ice-cold sterile water and the radioactivity was determined using a LKB Wallac 1211 rackbeta scintillation counter.
The N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM 4–64) (Molecular Probes) staining was performed according to Vida and Emr (1995). Briefly, the cells grown in YPD medium were harvested and resuspended to a density of OD600 2 in YPD medium, and incubated at the appropriate temperatures. The cells were harvested and resuspended in YPD medium containing 40 µM FM 4-64 to OD600 4, and incubated for 20 min at 24°C for internalization. The samples were harvested, resuspended to OD600 4 and incubated for 60 min at 24°C, and viewed. For surface labelling, the cells were incubated for 30 min on ice, placed immediately on slides and viewed. A Nikon Microphot-FXA microscope with a G-25 filter (510–560 nm) was used. Fluorescence exposures ranged from 1 to 6 s, DIC exposures were for 1–3 s, using Kodak Gold Ultra or Agfa HDC plus film (ASA 400). To immobilize the cells, 8 µl of a 1 mg ml−1 solution of concanavalin A in PBS was air-dried on 75 × 25 mm slides, 1–2 µl of cell suspension was applied and a 22 × 22 mm coverslip was gently layered on top of the cells. For immunofluorescence staining, the cells, grown in YPD medium to OD600 0.5–1, were fixed with 4% formaldehyde for 2 h at room temperature (RT) and transferred to 1.2 M sorbitol in 0.1 M KPO4 buffer (pH 6.8). β-Mercaptoethanol and lyticase (Sigma) were added to the final concentrations of 0.6% and 5 U ml−1, respectively, and the cells were incubated for 15 min at RT, washed twice with the sorbitol solution and placed on poly lysine-coated microscopic slides. The cells were permeabilized with ice-cold aceton (H984, β-lactamase antiserum) or methanol (others strains and antisera) for 5 min, air-dried, and treated for 30 min with PBS containing 1% milk powder, 0.1% BSA, 0.1% octyl glucoside and 0.02% NaN3. β-Lactamase antiserum (1:500) or Lhs1p antiserum (1:100; a kind gift of Dr E. Craig) were added and the samples incubated for 1 h at RT or overnight in the cold. The samples were washed eight times with the milk powder/BSA solution and rhodamine-conjugated anti-IgG (1:100) (Santa Cruz Biotechnology) was added. After 30–60 min, the cells were washed and viewed with an Olympus provis AX70 fluorescent microscope followed by digital photographing using Sensys photometrix.
Metabolic labelling and immunoprecipitation.
For metabolic labelling with [35S]-methionine/cysteine (1000 Ci mmol−1; Amersham) the yeast cells were grown in SC medium lacking methionine and cysteine, and labelled in the same medium (25 × 106 cells per 0.5 ml). Immunoprecipitation of lysed cell samples or medium samples was performed with β-lactamase antiserum (1:100) or CPY antiserum (1:100) and protein A-Sepharose for 2 h at 4°C, as described (Saris et al., 1997). For bulk protein synthesis experiments, the labelled cells were lysed, subjected to precipitation with 20% TCA in the cold and counted for precipitated radioactivity.
Next, 108 cells were harvested and resuspended to a density of OD600 8 in YPD medium, incubated at the appropriate temperatures, and fixed by addition of 6% paraformaldehyde and 4% glutaraldehyde in 0.167 M citrate phosphate buffer, pH 5.5, to the final concentrations of 3% and 2%, respectively, for 2 h at RT. The cells were post-fixed with 2% KMnO4 and dehydrated in a graded series of ethanol. The cells were embedded in Spurr's resin and thin-sectioned with Reichert-Jung Ultracut E ultramicrotome. The sections were stained with lead citrate and uranyl acetate and viewed with a Jeol JEM-1200EX instrument.
Other materials and methods
In the thermotolerance assay, cells were preincubated in Wassermann tubes for 1 h at 37°C before shifting them to 50°C. Duplicate samples of 2 × 106 cells were removed after 20 min, diluted and plated on YPD plates. The colonies were counted after 4 days of incubation at 24°C (Sanchez and Lindquist, 1990; Saris et al., 1997). Intra- and extracellular β-lactamase activities were determined as described before (Saris et al., 1997). SDS–PAGE was in 8% gels. Glucose consumption by the yeast cells was followed by determining its concentration in the medium of duplicate cell samples during incubations using the Gluco-quant kit of Boehringer.
This work was supported by the Academy of Finland (grants 38017 and 41409). M.M. is a Biocentrum Helsinki fellow. We thank Ms A. L. Nyfors and Ms M. Kankainen for technical assistance and Ms H. Ruoho for preparing the figures. Dr S. Lindquist, Dr J. Gancedo, Dr R. Hagenauer-Tsapis, Dr P. Silver and Dr R. Himmelreich generously provided yeast strains and plasmids and Dr E. Craig antiserum.