Effect of heat shock on synthesis and phosphorylation of nuclear and cytoplasmic proteins in the fungus Achlya

Abstract

Heat shock induced by an increase in temperature from 28 to 37 degrees C led to changes in synthesis and phosphorylation of cytoplasmic and nuclear proteins in the aquatic fungus Achlya. In the cytoplasmic fraction a marked increase in [35S]methionine labelling of proteins in the molecular weight range of 96 000, 85 000, 74 000, and 70 000 was observed. Two-dimensional electrophoresis resolved each of these classes of proteins into several components. Major changes in the nuclear fraction included the increased [35S]methionine labelling of 43 000 and 28 000-23 000 proteins. A marked decline in the synthesis of many other proteins was also evident. The heat-shock-induced changes in labelling patterns became evident as early as 20 to 60 min after treatment, but they were transient. With continued incubation at the heat-shock temperature, the cells appeared to adapt to the new temperature conditions. Both cytoplasmic and nuclear proteins returned to nearly normal labelling patterns within 100 to 140 min at 37 degrees C. Changes in phosphorylation of histone and nonhistone nuclear proteins were also noted. Achlya histone H3 and the putative oomycete-specific histone "alpha" appeared highly phosphorylated after heat shock. Since phosphorylation of histone H3 is primarily associated with chromatin condensation, it is possible that rapid chromatin condensation is an initial response to heat shock in Achlya.

Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.

“…Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.We have previously reported that in the aquatic fungus Achlya ambisexualis at least 10 molecular weight classes of proteins (96,000 [96K], 85K, 70K to 74K, 43K, 28K, 27K, 24K, 23K, and 18K) are synthesized within 20 to 60 min after a rapid elevation of temperature (heat shock) from 28 to 37°C (29,30). This heat shock response was accompanied by changes in the levels of phosphorylation of several nuclear proteins, including acid-insoluble proteins, as well as the apparent marked phosphorylation of two Achlya histones.…”

“…Before labeling, cells were collected by filtration, transferred to a phosphate-reduced (0.004 mM) mating medium described by Barksdale and Lasure (2) and allowed to equilibrate for 2 h. All cells were prelabeled at 28°C with 12 i,Ci of carrier-free 32P, per ml (New England Nuclear Corp.) for 30 min. Cells were then either maintained at 28°C for an additional 60 min (control) or subjected to a rapid elevation of the temperature to 37°C as described previously (29,30) and then maintained at 37°C for an additional 60 min (heat shock). Thus, the total labeling period in the presence of 32p; for both control and heat-shocked cultures was 90 min.…”

“…We have previously reported that in the aquatic fungus Achlya ambisexualis at least 10 molecular weight classes of proteins (96,000 [96K], 85K, 70K to 74K, 43K, 28K, 27K, 24K, 23K, and 18K) are synthesized within 20 to 60 min after a rapid elevation of temperature (heat shock) from 28 to 37°C (29,30). This heat shock response was accompanied by changes in the levels of phosphorylation of several nuclear proteins, including acid-insoluble proteins, as well as the apparent marked phosphorylation of two Achlya histones.…”

“…Cells of A. ambisexualis J. Raper E87 were grown as described elsewhere (29). Before labeling, cells were collected by filtration, transferred to a phosphate-reduced (0.004 mM) mating medium described by Barksdale and Lasure (2) and allowed to equilibrate for 2 h. All cells were prelabeled at 28°C with 12 i,Ci of carrier-free 32P, per ml (New England Nuclear Corp.) for 30 min.…”

“…For DNase I digestion studies, cells were grown in Barksdale mating medium (2) and labeled for 18 h with 1 ,uCi of [methyl-3H]thymidine per ml (New England Nuclear Corp.). Growth, heat shock conditions, and the nuclear isolation procedure were as described previously (29). Nuclei were purified from equal wet weights of control and heat-shocked cells, suspended in buffer (40 mM spermine, 1 mM spermidine, and 10% glycerol), and washed once.…”