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. 2005 Mar 15;386(Pt 3):433-43.
doi: 10.1042/BJ20041071.

Identification and characterization of the human ARD1-NATH protein acetyltransferase complex

Thomas Arnesen  1 , Dave Anderson, Christian Baldersheim, Michel Lanotte, Jan E Varhaug, Johan R Lillehaug
Affiliations

Identification and characterization of the human ARD1-NATH protein acetyltransferase complex

Thomas Arnesen et al. Biochem J. .

Abstract

Protein acetyltransferases and deacetylases have been implicated in oncogenesis, apoptosis and cell cycle regulation. Most of the protein acetyltransferases described acetylate epsilon-amino groups of lysine residues within proteins. Mouse ARD1 (homologue of yeast Ard1p, where Ard1p stands for arrest defective 1 protein) is the only known protein acetyltransferase catalysing acetylation of proteins at both alpha-(N-terminus) and epsilon-amino groups. Yeast Ard1p interacts with Nat1p (N-acetyltransferase 1 protein) to form a functional NAT (N-acetyltransferase). We now describe the human homologue of Nat1p, NATH (NAT human), as the partner of the hARD1 (human ARD1) protein. Included in the characterization of the NATH and hARD1 proteins is the following: (i) endogenous NATH and hARD1 proteins are expressed in human epithelial, glioma and promyelocytic cell lines; (ii) NATH and hARD1 form a stable complex, as investigated by reciprocal immunoprecipitations followed by MS analysis; (iii) NATH-hARD1 complex expresses N-terminal acetylation activity; (iv) NATH and hARD1 interact with ribosomal subunits, indicating a co-translational acetyltransferase function; (v) NATH is localized in the cytoplasm, whereas hARD1 localizes both to the cytoplasm and nucleus; (vi) hARD1 partially co-localizes in nuclear spots with the transcription factor HIF-1alpha (hypoxia-inducible factor 1alpha), a known epsilon-amino substrate of ARD1; (vii) NATH and hARD1 are cleaved during apoptosis, resulting in a decreased NAT activity. This study identifies the human homologues of the yeast Ard1p and Nat1p proteins and presents new aspects of the NATH and hARD1 proteins relative to their yeast homologues.

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Figures

Figure 1
Figure 1. hARD1 and NATH
(A) Alignment of yeast, mARD1 and hARD1. Identities are in black backgrounds and conservative substitutions are in grey backgrounds. (B) Schematic models (only showing sizes) of the 235 amino acids of hARD1 and the 866 amino acids of NATH. hARD1: acetyltransferase domain at amino acids 45–130 and the putative NLS at amino acids 78–83 are indicated. NATH: TPR motifs at amino acids 46–79, 80–113, 374–407, 408–441, and the putative NLS at amino acids 612–628 and a coiled-coil region at amino acids 583–635 are indicated.
Figure 2
Figure 2. Detection of endogenous NATH and hARD1
(A) Different cell types were lysed and analysed by SDS/PAGE and Western blotting. The membrane was incubated with a rabbit hARD1-peptide-specific polyclonal antibody. *, A slowly migrating hARD1 variant, a hARD1 containing complex or probably an unspecific band. Lanes: 1, ARO; 2, HEK-293; 3, HeLa; and 4, NB4. (B) As in (A) using an NATH-specific polyclonal antibody. The experiment was repeated more than three times.
Figure 3
Figure 3. Immunoprecipitation of NATH–hARD1 complexes
(A) HEK-293 cells stably expressing hARD1–V5 were harvested and the lysates were immunoprecipitated (IP) with the indicated antibodies (IgG denotes rabbit serum). The immunoprecipitates were analysed by SDS/PAGE and Western blotting. The membrane was incubated with anti-NATH. (B) A normal population of HEK-293 cells was immunoprecipitated and processed as in (A) with the addition of anti-hARD1. Experiments were repeated more than ten times.
Figure 4
Figure 4. The C-terminal of hARD1 is not involved in NATH–hARD1 interaction
(A) Some hARD1–V5 deletion proteins are outlined. The putative acetyltransferase motif (amino acids 45–130) is dotted, whereas the putative NLS (amino acids 78–83) is grey. (B) HEK-293 cells were transiently transfected with the indicated hARD1–V5 deletion plasmids. Cells were then harvested and the lysates were immunoprecipitated (IP) with anti-V5. The immunoprecipitates were analysed by SDS/PAGE and Western blotting. The membrane was incubated with anti-NATH and anti-V5. The experiment was repeated three times.
Figure 5
Figure 5. MS identification of proteins
HEK-293 cells stably expressing hARD1–V5 were harvested and lysates were immunoprecipitated and then subjected to serial lysC endoprotease/trypsin digestion and the peptides were identified by ion-trap MS. (A) MS/MS spectrum of the NATH-derived +2 peptide RLPLNFLSGEK, with the C-terminal y ions and N-terminal b ions labelled as assigned. All fragment ions had a +1 charge. The match between this spectrum and that predicted for the peptide had a cross-correlation coefficient (Xcorr) of 3.45. The y1 and b1 ions were below the low m/z detection limit in this experiment. The position of the fragmented parent ion is indicated by the double dagger on the x-axis for all three peptides. A second peptide, TQQTSPDKVDYEYSELLLYQNQVLR, sequenced separately twice, also indicated the presence of NATH in the affinity extract. (B) MS/MS spectrum of the hARD1 peptide AALHLYSNTLNFQISEVEPK, which was sequenced separately twice. This peptide had a charge of +2. The assigned y and b ion peaks (all +1) are indicated; the ions y1–y2 and b1–b3 were below the lower limit of detectable m/z in this experiment. The match between the predicted and observed spectrum was described by a very high Xcorr of 5.68. A second peptide assigned to hARD1 was MEEDPDDVPHGHITSLAVK. (C) MS/MS spectrum of the 60 S ribosomal protein P2-derived peptide YVASYLLAALGGNSSPSAK is shown, with the C-terminal y ions and N-terminal b ions labelled as assigned. All fragment ions had a +1 charge. The match between this spectrum and that predicted for the peptide had Xcorr of 4.29.
Figure 6
Figure 6. Detection of NATH, hARD1 and RACK1 in polysomal fractions
HEK-293 cells were treated with 20 μg/ml cycloheximide for 5 min before dithiobis(succinimidyl propionate) cross-linking and lysis. After an initial centrifugation, the CS was ultracentrifuged at 436000 g for 25 min through a 25% sucrose cushion, and the resultant supernatant (US) and P were analysed by SDS/PAGE and Western blotting. The membrane was incubated with the following antibodies: anti-NATH, anti-hARD1, anti-RACK1 and anti-P (ribosomal protein P0). Aliquots of the initial supernatant (S) and the US correspond to approx. 5% of the total material included in P. The experiment was repeated three times.
Figure 7
Figure 7. Localization of NATH and hARD1 by immunofluorescence
HeLa cells (A–D) and GaMg cells (E–H) were fixed and labelled with the indicated primary antibodies and thereafter with Alexa-488-conjugated anti-rabbit antibodies (green). Blue DAPI (4′,6-diamidino-2-phenylindole) staining (B, D, F, H) displays the nuclei of the cells. HeLa cells were transiently transfected with plasmids expressing NATH–V5 (I–L) and hARD1–V5 (M–P). Anti-V5 antibodies and Alexa-488 conjugated anti-mouse antibodies (green) were used to visualize NATH–V5 (I) and hARD1–V5 (M). Anti-NATH/anti-hARD1 and Alexa-568 conjugated antibodies (red) were used to visualize the endogenous hARD1 (J) and NATH (N). Nuclear DAPI staining is seen in blue (K, O). Green and red staining were overlaid (L, P). (Q–V) HeLa cells were co-labelled with anti-HIF1-α and anti-hARD1. Alexa-488 conjugated antibodies (green) were used to visualize hARD1 (Q, T) and Alexa-568 antibodies (red) to visualize HIF-1α (R, U). (S) DAPI-staining (blue) of cells in images (Q/R). (V) hARD1/HIF-1α (green/red) overlay of one HeLa nucleus from images (T/U). At least three independent experiments were performed.
Figure 8
Figure 8. Cleavage of NATH and hARD1 in apoptosis
(A) HeLa cells untreated (ctl.) or treated for 20 h with 10 μM daunorubicin (Dau). Shown in (A–D) are NATH, NATH-t, hARD1 and hARD1-t. (B) NB4 cells untreated (ctl.) or treated for 24 h with 200 nM Dau. % Apoptosis is the percentage of cells displaying apoptotic features as assessed by light microscopy. (C) NB4 cells untreated (0) or treated for 24 h with increasing concentrations of As2O3 as indicated. Bands representing hARD1-t and NATH-t are detected from 0.5 μM As2O3. Pictures display NB4 cells (100×) untreated, <3% apoptosis (left) or treated with 2 μM As0, approx. 50% apoptosis (right). (D) NB4 cells treated for 20 h with 200 nM Dau and for 10 h with 20 μM Z-VAD-FMK as indicated. (E) NATH–hARD1 complexes were immunoprecipitated from HeLa cells using limiting amounts of anti-hARD1 or rabbit IgG as a negative control. Apoptosis was induced by 10 μM daunorubicin for 20 h. The immunoprecipitates were incubated with corticotropin peptide amino acids 1–24 and [3H]acetyl-CoA, and the acetyl incorporation in corticotropin was determined by scintillation counting (c.p.m.). Percentage activity was adjusted according to the presence of hARD1/hARD1-t in the precipitates detected by Western blotting followed by densitometry. Five independent experiments were performed.
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