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Intracellular localization of Neurospora crassa endo-exonuclease and its putative precursor
Abstract:
Endo-exonuclease of rapidly growing mycelia of Neurospora crassa was found to be distributed in a ratio of about 1.6:1 in vacuoles and in mitochondria where it is associated with the inner membrane. Although the activity in vacuoles was readily released by osmotic shock, very little of that in mitochondria was released by this method. The mitochondrial activity was partially (60 to 70%) released by sonication, and the remaining activity was solubilized in the presence of Triton X-100. An inactive form of endo-exonuclease, activated in vitro by treatment with trypsin, is present in mycelia at a level over four times that of active enzyme. It was found to be distributed in a ratio of about 2.5:1 in the cytosol and in the inner membrane of mitochondria. The mitochondrial protein was more tightly bound than the active enzyme. Very little of the inactive enzyme was released by sonication, but it was solubilized in the presence of Triton X-100. The intracellular distribution of active and inactive forms of endo-exonuclease differs in a mutagen-sensitive mutant of Neurospora crassa (uvs-3) which shows many pleiotropic effects. The most striking difference in distribution is in the mitochondria where endo-exonuclease is present almost entirely in the inactive form at a level 30% higher than in wild-type mitochondria.
Insights
Neurospora crassa endo-exonuclease exists in active and inactive forms within cells, primarily in vacuoles, mitochondria, and cytosol. A mutant strain shows altered distribution, with inactive enzyme accumulating in mitochondria.
Area of Science:
- Molecular Biology
- Cell Biology
- Mycology
Background:
- Endo-exonuclease plays a role in DNA repair and replication.
- Understanding enzyme localization is crucial for elucidating its function.
- Neurospora crassa is a model organism for genetic and biochemical studies.
Purpose of the Study:
- To investigate the intracellular distribution of active and inactive endo-exonuclease in Neurospora crassa.
- To compare enzyme localization in wild-type and a mutagen-sensitive mutant (uvs-3).
- To characterize the release and solubilization properties of the enzyme from different cellular compartments.
Main Methods:
- Cell fractionation and differential centrifugation to isolate vacuoles, mitochondria, and cytosol.
- Enzyme activity assays to quantify active and inactive endo-exonuclease.
- Osmotic shock, sonication, and detergent treatment (Triton X-100) to assess enzyme release and solubilization.
- Comparison of enzyme distribution in wild-type and uvs-3 mutant strains.
Main Results:
- Active endo-exonuclease is found in vacuoles and mitochondria (inner membrane) at a 1.6:1 ratio.
- Inactive endo-exonuclease, activated by trypsin, is present in cytosol and mitochondria (inner membrane) at a 2.5:1 ratio, at levels fourfold higher than active enzyme.
- Mitochondrial-bound enzyme, especially the inactive form, is more tightly associated and less readily released than vacuolar or cytosolic forms.
- The uvs-3 mutant exhibits altered distribution, with a significant increase in inactive endo-exonuclease within mitochondria.
Conclusions:
- Neurospora crassa possesses both active and inactive forms of endo-exonuclease with distinct intracellular localizations.
- Mitochondrial association, particularly of the inactive form, suggests specific regulatory mechanisms.
- The altered distribution in the uvs-3 mutant highlights the enzyme's role in DNA repair pathways and potential pleiotropic effects of mutations affecting this process.