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bioRxiv · 10.1101/814327

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+ biosynthetic enzymes in Saccharomyces cerevisiae

Croft, T.·Venkatakrishnan, P.·James Theoga Raj, C.·Groth, B.·Cater, T.·Lin, S.-J.
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Abstract

NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are not completely understood. We previously identified the N-terminal (Nt) protein acetyltransferase complex NatB as a NAD+ homeostasis factor. Cells lacking NatB show an approximate 50% reduction in the NAD+ level and aberrant metabolism of NAD+ precursors, which are associated with a decrease of nicotinamide mononucleotide adenylyltransferases (Nmnat) protein levels. Here we show this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins, and not other NatB substrates. Nt-acetylation is a critical regulator of protein degradation by the N-end rule pathways, indicating absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t1/2) of non-Nt-acetylated Nmnats does not significantly differ from Nt-acetylated Nmnats, suggesting reduced Nmnat levels in NmatB mutants are not due to increased post-translational degradation of non-Nt-acetylated Nmnats. In line with these observations, deletion or depletion of N-rule pathway ubiquitin E3 ligases in NatB mutants is not sufficient to restore NAD+ levels. Moreover, the status of Nt-acetylation does not alter the rate of translation initiation of Nmnats. Collectively our studies suggest absence of Nt-acetylation may increase co-translational degradation of nascent Nmnat polypeptides, which results in reduced Nmnat levels in NatB mutants. Nmnat activities are essential for all routes of NAD+ biosynthesis. Understanding the regulation of Nmnat protein homeostasis will facilitate our understanding of the molecular basis and regulation of NAD+ metabolism.

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Croft, T., Venkatakrishnan, P., James Theoga Raj, C., Groth, B., Cater, T., Lin, S.-J.. 2019-10-29. N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+ biosynthetic enzymes in Saccharomyces cerevisiae. https://doi.org/10.1101/814327

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