Neurospora endo-exonuclease in heat-shocked mycelia: evidence for a novel heat shock induced function

Shifting rapidly growing mycelial cultures of Neurospora crassa from 30 to 45 °C for 2 h resulted in 75% of the active endo-exonuclease, an enzyme implicated previously in recombinational DNA repair, being released from nuclei and vacuoles into the cytosol, where it was inactivated by a heat shock induced inhibitor. Cycloheximide, at a level which inhibited [35S]methionine incorporation into the protein of unshocked cells by 95%, did not block the release of endo-exonuclease from the organelles during heat shock, but rather allowed a 6- to 15-fold accumulation of the nuclease activity in the cytosol. The heat shock also resulted in depletion of about 65% of nuclear, and over 35% of cytosolic, trypsin-activable endo-exonuclease (comprised of both endo-exonuclease–inhibitor complex and endo-exonuclease precursor?). Heat shock also caused the release of about 40% of the total trypsin-activable endo-exonuclease plus active enzyme from mitochondria. During a subsequent 1-h recovery at 30 °C, trypsin-activable endo-exonuclease, but not active enzyme, was restored in full to the nuclei. Some restoration of vacuolar endo-exonuclease was also observed. In the cytosol, neither the level of active enzyme nor the level of trypsin-activable endo-exonuclease was appreciably restored during this recovery period. The accumulation of the new inhibitor in the cytosol of heat-shocked mycelia was estimated to be 20-fold. Although cycloheximide prevented this accumulation, it is not clear whether the inhibitor itself is synthesized in response to heat shock or whether it is generated from a preexisting form by some other heat shock induced function. The high level of inhibitor that accumulated during heat shock persisted throughout the 1-h recovery period. The properties of this inducible inhibitor showed both similarities to and differences from a highly specific constitutive inhibitor of endo-exonuclease purified from unshocked mycelia.Key words: Neurospora crassa, endo-exonuclease, heat shock, inducible, inhibitor.