Academic journal article Genetics

Nonself Recognition through Intermolecular Disulfide Bond Formation of Ribonucleotide Reductase in Neurospora

Academic journal article Genetics

Nonself Recognition through Intermolecular Disulfide Bond Formation of Ribonucleotide Reductase in Neurospora

Article excerpt

ABSTRACT Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N. crassa results in a high molecular weight UN-24 protein complex. A 63-amino-acid portion of the C terminus was sufficient for un-24^sup PA^ incompatibility activity. Redox active cysteines that are conserved in type I RNRs and essential for their catalytic function were found to be required for incompatibility activity of both UN-24^sup OR^ and UN-24^sup PA^. Our results suggest a plausible model of un-24 incompatibility activity in which the formation of a complex between the incompatible RNR proteins is potentiated by intermolecular disulfide bond formation.

HYPHAL fusion between conspecific fungi can result in the formation of heterokaryons: cells with more than one nuclear type. Growth of heterokaryotic cells is governed by nonself recognition loci (Glass and Kaneko 2003; Smith and Lafontaine 2013) that function to restrict transfer of parasitic genetic elements (Biella et al. 2002). Neurospora crassa has 11 heterokaryon incompatibility loci that regulate nonself recognition during vegetative growth (Perkins 1988). Allelic differences at one or more of these loci triggers incompatibility activity leading to cell death (Jacobson et al. 1998).

The N. crassa incompatibility locus un-24 contains two genes, un-24 and het-6, that function together as an incompatibility gene complex (Lafontaine and Smith 2012). Heteroallelism at un-24 results in an incompatibility reaction in strains in which het-6 is deleted, indicating that un-24 can autonomously mediate nonself recognition. un-24 also encodes the large subunit of a type I ribonucleotide reductase (RNR) and is therefore the only described example of an RNR large subunit having nonself recognition function (Smith et al. 2000b). RNR is an essential enzyme that reduces ribonucleotides to their corresponding deoxyribonucleotides and is necessary for DNA synthesis and repair (Elledge et al. 1993). Type I RNRs are found in all eukaryotic species, as well as selected viruses and prokaryotes. The holoenzyme functions as a tetramer composed of two large subunits (R1) and two small subunits (R2) (Reichard 1993). R1 contains the catalytic site and two allosteric effector sites (Eriksson et al. 1997; Reichard 2002). The R2 subunit contains a dinuclear iron center that generates a tyrosil radical (Y122 in Escherichia coli) (Nordlund and Eklund 1993). Based on studies done in E. coli, the reaction mechanism of RNR involves five conserved cysteines (C225, C439, C462, C754, and C759) and a radical intermediate (Aberg et al. 1989; Mao et al. 1992). The cysteine residues located in the catalytic site (C225, C439, and C462) mediate reduction of the nucleotide via disulfide bond formation. Subsequently, the active site disulfide bond is transferred to C754 and C759 of the C terminus region, which is then reduced by glutaredoxin or thioredoxin to regenerate the enzyme (Aberg et al. 1989; Mao et al. 1992). Although the mechanism by which this disulfide bond transfer occurs is unknown, it has been demonstrated that, in yeast, the labile structure of the C terminus allows for the C-terminal cysteine residues of one R1 subunit to act in trans to reduce the active site of the neighboring R1 subunit (Zhang et al. 2007).

The tertiary structure of the large RNR subunit from Saccharomyces cerevisiae (Rnr1p) is similar to the E. coli ortholog (Xu et al. 2006), while the predicted protein sequence of UN-24 in N. crassa is ~85% similar to Rnr1p. A major departure from the similarity of these proteins occurs in the C terminus (Smith et al. …

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