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Expression, localization, and biochemical characterization of nicotinamide mononucleotide adenylyltransferase 2 - PubMed

  • ️Fri Jan 01 2010

Expression, localization, and biochemical characterization of nicotinamide mononucleotide adenylyltransferase 2

Paul R Mayer et al. J Biol Chem. 2010.

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.

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Figures

FIGURE 1.
FIGURE 1.

Nmnat2 is a neuronal protein and expression is developmentally regulated. A, to assess the specificity of an in-house antibody targeted against full-length Nmnat2, Neuro-2a, a neuroblastoma cell line reported to express Nmnat2 transcript (BioGPS), were infected with lentiviruses (MOI of 1) expressing either shRNA against Nmnat2 (lanes A and B) or EGFP (lane C). Total lysate from each was compared by Western blot. B, to evaluate the tissue expression of Nmnat2, lysate from various tissues were analyzed by Western blot. Note that a slightly slower migrating band is observed in several tissues including brain, marked with an asterisk; this band is nonspecific as shown in C. C, comparison of brain lysate from mice in which both Nmnat2 alleles have been trapped (Tr/Tr) versus wild-type controls (+/+). Each lane represents a single animal. As expected (see text), Nmnat2 expression in gene trap mouse brain is ∼50% of wild-type controls. Note the same nonspecific band observed in B, marked with an asterisk. D, to evaluate the cellular expression of Nmnat2, lysates from various cell types were analyzed by Western blot. Nmnat2 is detected in a human neuroblastoma cell line SH-SY5Y, but not in primary glia, U87 or U343, which are glioblastoma cell lines, or either of the negative controls NIH3T3 or HEK293T. In SH-SY5Y, Nmnat2 is dramatically up-regulated upon differentiation. E, Nmnat2 expression increases during maturation of primary cortical neurons similarly to synaptobrevin 2, a critical synaptic vesicle protein. F, expression of Nmnat2 during mouse development. Mouse brain lysate was obtained from a pair of male and female mice at birth and every 4 days thereafter as indicated. For each panel, an equal amount of protein was loaded in each lane; actin, GAPDH, or VCP are used as loading controls. An asterisk indicates a nonspecific band. NF, neurofilament; Syb 2, synaptobrevin 2; DIV, days in vitro; Diff., differentiated; Undiff., undifferentiated. HC, heavy chain.

FIGURE 2.
FIGURE 2.

Residues 143–169 are necessary for Golgi localization and membrane association. A, schematic of human NMNAT2 (NP_055854) indicates residues that have been deleted in a given mutant. A characteristic stretch of residues poorly conserved between Nmnat1, -2, and -3, referred to as the LHD, is illustrated with a dotted line; conserved regions are in solid yellow. B, HeLa cells infected with lentiviruses (MOI ∼ 3) expressing either Nmnat2 or the indicated mutant (with EGFP tag) were fixed and then probed for GM130 to assess Golgi localization. Scale bar, 10 μm.

FIGURE 3.
FIGURE 3.

Nmnat2 is a palmitoylated peripheral membrane protein. A, sequence alignment reveals Cys164 and Cys165 as potential targets of palmitoylation. Palmitoylation lacks a well defined motif but occurs on cysteines frequently with nearby basic residues. Cys164 and Cys165 are highlighted in red, whereas nearby conserved basic residues are in boldface italic. B, HEK293T were transfected and metabolically labeled with [3H]palmitate. Following immunoprecipitation, incorporation of radiolabel was assessed by in-gel fluorography. To assess the efficiency of immunoprecipitation, a 1:10 dilution of eluted protein was analyzed by Western blot (WB). CC, Cys164 and Cys165; SS, Ser164 and Ser165. C, HeLa were infected with lentiviruses (MOI of 3) expressing either Nmnat2-EGFP, Nmnat2 C164S/C165S-EGFP, or EGFP and then fixed and probed for GM130, a well characterized cis-Golgi marker (36). D, infected HeLa cells were permeabilized with digitonin prior to fixation to assess the solubility of wild-type Nmnat2 and C164S/C165S mutant. Note that under these conditions, the nuclear membrane remains intact. Scale bar, 10 μm.

FIGURE 4.
FIGURE 4.

Biochemical characterization of Nmnat2 membrane association. A, mouse brains were homogenized and fractionated in detergent-free buffer as described under “Experimental Procedures.” An asterisk indicates a nonspecific band. B, to characterize the nature of the interaction between Nmnat2 and the membrane, P100 pellets were extracted with either high salt (1

m

NaCl), a chemical denaturant (4

m

urea), or a selection of detergents: 1% CHAPS (zwitterionic), 1% OG (nonionic), or 1% LDS (anionic). P and S refer to pellet and supernatant collected after a 30-min extraction on ice and a subsequent 100,000 × g spin. C, Nmnat2 is efficiently extracted in 2% Triton X-114 and partitions with detergent following phase separation, as expected for a palmitoylated protein.

FIGURE 5.
FIGURE 5.

Nmnat2 low homology domain is insufficient for stable interaction with Golgi. A, HeLa cells infected with lentivirus (MOI ∼ 3) expressing LHD(Nmnat2)-EGFP were fixed and probed for GM130. B, treatment of these cells with a proteasome inhibitor, MG132 (20 μ

m

for 2.5 h), boosts the overall intensity of fluorescent signal revealing weak colocalization between LHD(Nmnat2)-EGFP and GM130 (arrows). Images were taken using the same exposure settings for both dimethyl sulfoxide (DMSO, negative control) and MG132-treated cells. Scale bar, 10 μm.

FIGURE 6.
FIGURE 6.

Exogenous Nmnat2 is toxic to primary neurons. A, primary cortical neurons were infected with lentivirus expressing either Nmnat2-FLAG or Nmnat2 C164S/C165S-FLAG. Approximately 4 days after infection, cultures were fixed and probed with an anti-FLAG antibody. Nmnat2, but not Nmnat2 C164S/C165S, is toxic to neurons, which results in massive neuronal death; arrows indicate neurons shown in the inset. For comparison, a glial cell in each image is marked with an asterisk. Scale bar, 50 μm. B, primary cortical neurons were infected with lentivirus (MOI ∼ 3) expressing Nmnat2-EGFP. Nmnat2 localizes to Golgi (as in HeLa cells) but also dendrites and axons (scale bar, 10 μm). Shown is a magnified view of a dendrite (lower panel; scale bar, 2 μm).

FIGURE 7.
FIGURE 7.

Endogenous Nmnat2 localizes to synaptic terminals. A, schematic of synaptosome purification protocol. Synaptosomes are detached nerve terminals formed during homogenization and contain membranes derived from both pre- and postsynaptic compartments. For P1, S1, P11, S11, P200, and S200, P refers to pellet, and S refers to supernatant, whereas numbers refer to the force of centrifugation for each step (e.g. S200, supernatant resulting from 200,000 × g spin). B, Western blot analysis of fractionated mouse brains; an equal amount of protein was loaded for each sample. An asterisk indicates a nonspecific band. NRX, neurexin; Syb 2, synaptobrevin 2; synap., synaptosome; SV, synaptic vesicles; SPM, synaptic plasma membrane; mito, mitochondria.

FIGURE 8.
FIGURE 8.

Nmnat2 localizes to Rab7 late endosomes and trans-Golgi network in HeLa cells. HeLa cells were infected with lentiviruses (MOI of 3) expressing Nmnat2-EGFP and then fixed and probed for Rab7, a late endosome marker (67) (top panel); TGN46, a trans-Golgi network marker (55) (middle panel); or EEA1, an early endosome marker (56) (bottom panel). Dotted lines indicate regions in the cytoplasm where Rab7 and Nmnat2 are closely associated. Arrows indicate where Nmnat2 does not co-localize with Rab7. Arrowhead indicates specific vesicle where Nmnat2 colocalizes with Rab7. An asterisk marks the nucleus in each cell. Scale bar, 10 μm.

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