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 mutagensensitive 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.

Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos

“…Like mitochondrial nucleases isolated from other organisms Chow and Fraser, 1983;Rosamond, 1981;Morosoli and Lusena, 1980;von Tigerstrom, 1982;Fraser and Cohen, 1983), this enzyme exhibits an absolute requirement for Mg" or Mn2+ which cannot be replaced by Ca2+ or Zn2+. In further analogy to enzymes isolated from other organisms, this nuclease produces 3' OH termini which can be extended by E. coli DNA polymerase I.…”

“…Although there are mutants available in fungi that have been associated with altered levels of mitochondrial nuclease (Fraser and Cohen, 1983;Chow and Resnick, 1987), they have not provided a full understanding of the function of those enzymes. Accordingly, we have undertaken the analysis of a mitochondrial nuclease in Drosophilu with thc ultimate goal of obtaining potentially more informative mutations in a multicellular organism.…”

“…It has been suggested that mitochondrial nucleases might be involved in DNA replication, repair and recombination. Fraser (1979), Fraser and Cohen (1983) and more recently Tomkinson et al (1988Tomkinson et al ( ,1990 have described a mitochondrial endonuclease activity specific for apurinic/apyrimidinic sites in DNA from mouse cells and a mammalian mitochondria1 ultraviolet endonuclease activity specific for ultraviolet-irradiated DNA. Such observations suggest that these nucleases might be involved in DNA repair or the latter enzyme might act to mark damaged mitochondrial genomes for subsequent degradation.…”

Identification and characterization of a mitochondrial endonuclease from yeast, Schizosaccharomyces pombe

“…mammalian enzymes were, identified in the mitochondria of Neurospora crassa (6,7,8), Aspergillus nidulans (9), and Saccharomyces cerevisiae (10,11). The endo-exonuclease activities of N. crassa were found in mitoehondria, nuclei, and vacuoles, and they are immunologicaUy related to each other (7).…”

Aurintricarboxylic acid as a nuclease inhibitor in fungal protoplasts

“…In fungal species, nuclease activity [6,7] must be controlled to improve the transformation systems. Nuclease-deficient mutants have been used, but without success [8].…”