In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone - PubMed (original) (raw)

In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone

D F Nathan et al. Proc Natl Acad Sci U S A. 1997.

Abstract

In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold. A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear. In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function. We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo. The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations. Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated. Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted.

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Figures

Figure 1

Figure 1

Role of Hsp90 in de novo protein folding of test substrates. Activity and accumulation of newly synthesized proteins were assayed in wild-type (W) or hsp90G170D mutant (M) cells as follows: p60v-src (A), β-galactosidase (B), firefly luciferase (C), bacterial luciferase-V.h.MAV (D), and bacterial luciferase-V.h.CTP5 (E). Cells grown to midlogarithmic phase at 25°C in glucose were shifted to galactose at the indicated temperatures to simultaneously induce the expression of test substrates and inactivate Hsp90G170D. At 25°C, activities in wild-type and hsp90G170D cells were similar, but they varied somewhat in individual cultures grown from different colonies of each strain. Values at higher temperatures (means and standard deviations) are therefore normalized to the activity of each culture at 25°C. For each substrate, at least three independent experiments were performed, each including three independent transformants of each strain. Boxed data below activity measurements show the accumulation of substrate proteins as determined by immunological reaction with specific antibodies (Western blotting). Equal loading was confirmed by probing blots with antibody specific for the yeast ribosomal protein L3. C indicates wild-type cells lacking a substrate expression plasmid. Loss of Hsp90 function does not affect the maturation or accumulation of β-galactosidase, and it affects the accumulation of firefly luciferase only at temperatures close to the denaturation temperature of the protein, but it strongly affects the maturation of a bacterial luciferase protein that has difficulty folding even in wild-type cells.

Figure 2

Figure 2

The absence of Hsp90 function does not affect the solubility of most of newly synthesized yeast proteins. Cells grown to mid-logarithmic phase at 25°C were labeled with [35S]methionine at 25°C, 34°C, or 36°C for 45 min and maintained at the same temperatures for an additional hour in unlabeled medium. Solubility of proteins was assessed in wild-type (W) and hsp90G170D (M) cells by centrifugation of cell lysates at 340,000 × g, followed by SDS/PAGE of total lysates (L), supernatants (S), and pellets (P). Proteins were visualized by autoradiography.

Figure 3

Figure 3

The absence of Hsp90 function has little effect on the heat-induced inactivation of test substrates. Wild-type (□) and hsp90G170D (⋄) cells expressing firefly luciferase (A) or bacterial luciferase-V.h.MAV (B) were grown to mid-logarithmic phase in galactose at 25°C to allow the accumulation of active test substrates. Cells were then shifted to the indicated temperatures for 15 min and the extent of enzyme inactivation was determined immediately. Enzyme activities were normalized to the activity present in each strain prior to heat inactivation. Data are the means and standard deviations of three independent experiments using three independent transformants.

Figure 4

Figure 4

Hsp90 speeds the recovery of heat-inactivated firefly luciferase. Firefly luciferase was inactivated at 42°C as described in the legend of Fig. 3. With the addition of a protein synthesis inhibitor, cells were returned to 34°C (a temperature at which most Hsp90 activity is lost in the hsp90G170D mutant). The recovery of previously synthesized, heat-inactivated firefly luciferase was monitored in wild-type (□) and hsp90G170D (⋄) cells as described in the text. Enzyme activities were normalized to the activity present in each strain prior to heat inactivation. Data are the means and standard deviations of three independent experiments using three independent transformants.

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