Expression, Localization, and Biochemical Characterization of Nicotinamide Mononucleotide Adenylyltransferase 2

Paul R Mayer,Nian Huang,Colleen M Dewey,Daniel R Dries,Hong Zhang,Gang Yu

doi:10.1074/jbc.m110.178913

Paul R Mayer, Nian Huang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.178913

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Abstract

Nicotinamide mononucleotide (NMN) adenylyltransferase 2 (Nmnat2) catalyzes the synthesis of NAD from NMN and ATP. The Nmnat2 transcript is expressed predominately in the brain; we report here that Nmnat2 is a low abundance protein expressed in neurons. Previous studies indicate that Nmnat2 localizes to Golgi. As Nmnat2 is not predicted to contain a signal sequence, lipid-binding domain, or transmembrane domain, we investigated the nature of this interaction. These experiments reveal that Nmnat2 is palmitoylated in vitro, and this modification is required for membrane association. Surprisingly, exogenous Nmnat2 is toxic to neurons, indicating that protein levels must be tightly regulated. To analyze Nmnat2 localization in neurons (previous experiments relied on exogenous expression in HeLa cells), mouse brains were fractionated, showing that Nmnat2 is enriched in numerous membrane compartments including synaptic terminals. In HeLa cells, in addition to Golgi, Nmnat2 localizes to Rab7-containing late endosomes. These studies show that Nmnat2 is a neuronal protein peripherally attached to membranes via palmitoylation and suggest that Nmnat2 is transported to synaptic terminals via an endosomal pathway.

Highlights

In addition to its role as a cofactor, NAD is a substrate for numerous enzymes including sirtuins, poly(ADP-ribose) polymerases, and ADP-ribosyl cyclases (e.g. CD38) [11]
Biochemical analysis shows that endogenous Nmnat2 behaves like a membrane protein but is difficult to solubilize suggesting that 1) palmitoylation directs Nmnat2 to detergent resistant membranes or 2) in addition to palmitoylation, Nmnat2 is held at the membrane via protein-protein interaction
We looked for cell lines expressing high levels of Nmnat2 transcript (BioGPS)

Summary

Introduction

In addition to its role as a cofactor, NAD is a substrate for numerous enzymes including sirtuins, poly(ADP-ribose) polymerases, and ADP-ribosyl cyclases (e.g. CD38) [11]. We have first sought to understand how Nmnat2 interacts with the Golgi, its cellular and developmental protein expression, and its localization in brain cells. We report here that Nmnat2 is a developmentally regulated, low abundance neuronal protein that localizes to Golgi and to vesicles and synaptic compartments.

Results

Conclusion