NAD(+), an essential molecule involved in a variety of cellular processes, is synthesized through de novo and salvage pathways. NAD(+) synthetase catalyses the final step in both pathways. Here we show that this enzyme is encoded by the QNS1 gene in Saccharomyces cerevisiae. Expression of Escherichia coli or Bacillus subtilis NAD(+) synthetases was able to suppress the lethality of a qns1 deletion, while a B. subtilis NAD(+) synthetase mutant with lowered catalytic activity was not. Overexpression of QNS1 tagged with HA led to elevated levels of NAD(+) synthetase activity in yeast extracts, and this activity can be recovered by immunoprecipitation using anti-HA antibody. An allele of QNS1 was constructed that carries a point mutation predicted to reduce the catalytic activity. Overexpression of this allele, qns1(G521E), failed to elevate NAD(+) synthetase levels and qns1(G521E) could not rescue the lethality caused by the depletion of Qns1p. These results demonstrate that NAD(+) synthetase activity is essential for cell viability. A GFP-tagged version of Qns1p displayed a diffuse localization in both the nucleus and the cytosol. Finally, the rat homologue of QNS1 was cloned and shown to functionally replace yeast QNS1, indicating that NAD(+) synthetase is functionally conserved from bacteria to yeast and mammals.

• PMID: 12898714
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Journal Article

Authors

Suda Y, Tachikawa H, Yokota A, Nakanishi H, Yamashita N, Miura Y, Takahashi N

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