Knockdown of human N alpha-terminal acetyltransferase complex C leads to p53-dependent apoptosis and aberrant human Arl8b localization

Abstract

Protein N(alpha)-terminal acetylation is one of the most common protein modifications in eukaryotic cells. In yeast, three major complexes, NatA, NatB, and NatC, catalyze nearly all N-terminal acetylation, acetylating specific subsets of protein N termini. In human cells, only the NatA and NatB complexes have been described. We here identify and characterize the human NatC (hNatC) complex, containing the catalytic subunit hMak3 and the auxiliary subunits hMak10 and hMak31. This complex associates with ribosomes, and hMak3 acetylates Met-Leu protein N termini in vitro, suggesting a model in which the human NatC complex functions in cotranslational N-terminal acetylation. Small interfering RNA-mediated knockdown of NatC subunits results in p53-dependent cell death and reduced growth of human cell lines. As a consequence of hMAK3 knockdown, p53 is stabilized and phosphorylated and there is a significant transcriptional activation of proapoptotic genes downstream of p53. Knockdown of hMAK3 alters the subcellular localization of the Arf-like GTPase hArl8b, supporting that hArl8b is a hMak3 substrate in vivo. Taken together, hNatC-mediated N-terminal acetylation is important for maintenance of protein function and cell viability in human cells.

FIG. 1.

Alignment of Mak3p and Mak31p homologues. Identities are given in dark gray. Conservative substitutions are given in light gray. (A) Putative homologues of S. cerevisiae (Sc) Mak3p: human (Hs) Nat12, Mus musculus (Mm) Nat12, S. pombe (Sp) NP_596246, D. melanogaster (Dm) NP_181348, A. thaliana (At) NP_181348, C. elegans (Ce) NP_504411. (B) Putative homologues of yeast Mak31p: Hs Lsm8, Hs Lsmd1, Sp NP588509, Dm NP_647660, M. sativa (Ms) P24715, At NP_187757, Thermoplasma acidophilum (Ta) NP_394385, Ce NP_498708. The alignments were performed using JalView (14) and MAFFT sequence aligner (22).

FIG. 2.

hMak10, hNat12/hMak3, and Lsmd1/hMak31 interact to form a stable complex. (A) HeLa cells expressing hMak10-V5 and Xpress-lacZ (negative control), Xpress-hNat12, Xpress-hNat5, Xpress-Lsm8, or Xpress-Lsmd1 were harvested and the lysates were immunoprecipitated (IP) with anti-Xpress antibody (Invitrogen). Immunoprecipitated proteins were identified by Western blotting using the indicated antibodies. (B) Cells expressing hMak10-V5, Lsmd1-V5, and Xpress-hNat12 or Xpress-lacZ (negative control) were immunoprecipitated as for panel A using an anti-Xpress antibody in the immunoprecipitation. Molecular mass (in kDa) is indicated on the left-hand side. Results shown are representative of more than three independent experiments.

FIG. 3.

Subcellular localization of hMak10-V5, Xpress-hMak3, and Xpress-hMak31. HeLa cells were transfected with hMak10-V5 (A and B), Xpress-hMak3 (C and D), or Xpress-hMak31 (E and F). Anti-V5 antibodies and Alexa-594-conjugated anti-mouse antibodies were used to visualize hMak10-V5 (A). Anti-Xpress antibodies and Alexa-488-conjugated anti-mouse antibodies were used to visualize Xpress-hMak3 (C). Anti-Xpress antibodies and Texas Red-conjugated anti-mouse antibodies were used to visualize Xpress-hMak31 (E). 4′,6-diamidino-2-phenylindole staining was used to visualize the nuclei of the cells (B, D, and F). Bar, 25 μm.

FIG. 4.

hNatC subunits cosediment with polysomal fractions in a salt-sensitive manner. (A) Polysomal pellets from HeLa cells expressing hMak10-V5, Xpress-hMak3, and Xpress-hMak3 were resuspended in buffer containing increasing concentrations of KCl. Cell lysate (L), supernatant post-first ultracentrifugation (S), and polysomal pellets after KCl treatment were analyzed by SDS-PAGE and Western blotting. The membrane was incubated with anti-V5, anti-Xpress, anti-L26 (ribosomal protein), and anti-CytC antibodies. Molecular mass markers (in kDa) are indicated on the left-hand side. (B) For endogenous hMak3, untransfected cells were treated as described for panel A and analyzed with anti-hMak3 antibody. Molecular mass (in kDa) is indicated on the left-hand side. Results shown are representative of more than three independent experiments.

FIG. 5.

hMak3 displays sequence-specific N-acetyltransferase activity. E. coli-expressed and purified MBP-hMak3 was analyzed for N^α-acetyltransferase activity using peptides differing in their N-terminal residues and [1-¹⁴C]acetyl coenzyme A. The acetyl incorporation was determined by isolation of the peptides followed by scintillation counting. Experiments were performed three to five times for each peptide. Error bars indicate standard deviations.

FIG. 6.

hMAK3, hMAK10, or hMAK31 knockdown induces apoptosis in HeLa cells. (A) Cells cultured in six-well plates were transfected with 50 nM hMAK3, hMAK10, or hMAK31 SMART pool siRNAs or control siRNA (siGAPDH or nontargeting siRNA). After 48 h, total RNA was isolated and processed by RT-PCR with specific primers against the genes of interest. (B) At 48 h posttransfection the cell proliferation rate was determined using a BrdU assay, measuring the amount of bromodeoxyuridine incorporated into nuclear DNA. The results are given as percent mitogenic activity. Error bars (B and C) represent standard deviations. P values for independent t tests for samples versus control are indicated (*, P < 0.001). (C) At 72 h posttransfection the cell viability was measured using a WST assay. The results are given as percent cell viability. P values for independent t tests for samples versus control are indicated (*, P < 0.00001). (D) Live cell imaging of hMAK3 knockdown cells 72 h posttransfection. Arrowheads indicate apoptotic cells. Hoechst 33342 staining was used to stain the nuclei. Phase contrast (PH) was used to visualize cells. (E) PARP cleavage was observed by harvesting cells 72 h posttransfection and analyzing cell lysates by Western blotting. The membrane was incubated in anti-cleaved PARP and anti-β-tubulin (loading control). All experiments were performed a minimum of three times. (F) At 72 h posttransfection hMAK3, hMAK10, or hMAK31 knockdown cells were analyzed for DNA breaks by using a TUNEL assay. Blue Hoechst 33342 staining was used to visualize the nuclei. Cells were visualized by phase contrast (PH).

FIG. 7.

Cell cycle analysis of hMAK3, hMAK10, and hMAK31 knockdown cells. Flow cytometric cell cycle analysis results are shown for hMAK3, hMAK10, and hMAK31 knockdown cells at 72 h posttransfection. Experiments were performed three times, and representative values are given.

FIG. 8.

hMAK3 knockdown induces apoptosis via p53 stabilization and transcriptional activation. (A) HeLa cells cultured in six-well plates were transfected with 50 nM hMAK3-1 or hMAK3-2 taken from the SMART pool sihMAK3 or with control siRNA (nontargeting siRNA). Daunorubicin (Dau) treatment was used as a positive control for apoptosis. At 72 h posttransfection, cell lysates were analyzed by Western blotting. The membrane was incubated with anti-cleaved α-Fodrin (Asp1185), anti-cleaved PARP (Asp214), anti-Mdm2, anti-p53, anti-phospho-p53 (Ser15), anti-phospho-p53 (Ser37), anti-hMak3, and anti-β-tubulin (loading control). Experiments were performed a minimum of three times, and representative results are shown. (B) Cells were treated as for panel A, but after harvesting, total RNA was isolated and processed by quantitative RT-PCR with gene-specific primers against hMAK3, KILLER/DR5, FAS, and NOXA. Error bars indicate standard deviations. P values for independent t tests for samples versus control are indicated (*, P < 0.0001). Values indicated in the diagram are given in the table below.

FIG. 9.

Expression of exogenous hMak3 rescues hMAK3 knockdown phenotypes. (A) The viability of hMAK3 knockdown cells expressing exogenous hMak3-V5 was compared to hMAK3 knockdown cells not expressing exogenous hMak3. Cell viability was measured 72 h posttransfection using a WST-1 assay. Results are given as percent cell viability. Experiments were performed in two parallel assays using two individual hMAK3 siRNAs. DharmaFECT Duo transfection reagent was used for cotransfection. Nontargeting SmartPool siRNA and lacZ-V5 were used as cotransfection controls. HeLa cells were used in all experiments. P values for hMak3 knockdown rescued with exogenous expression of hMak3 obtained with paired t tests are indicated: *, P < 0.004; **, P < 0.001. (B) Cells were treated as for panel A, and cell lysates were analyzed by SDS-PAGE and Western blotting. The membrane was incubated with anti-cleaved PARP (Asp214), anti-p53, anti-phospho-p53 (Ser37), anti-hMak3, anti-V5, and anti-β-tubulin (loading control). Results shown are representative of three independent experiments.

FIG. 10.

hMAK3 knockdown alters hArl8b localization. hArl8b-GFP was transfected into siControl cells (A to D) and sihMAK3 cells (E to H). (A and E) Localization of hArl8b-GFP (green); (B and F) the lysosomal marker LAMP-1 (red). (C and G) An overlay of hArl8b-GFP and LAMP-1, in addition to blue Hoechst 33342 staining for visualization of DNA. (D and H) Detailed view from panels C and F, respectively, as indicated by the marked areas in panels C and F. Bar, 25 μm. (I) hArl8b-GFP-transfected cells were counted (A to H), and the percentage of cells displaying a nonpunctuate distribution, as exemplified in panels E to H, was calculated. At least 500 transfected cells of each type from three independent experiments were registered. Error bars indicate the standard deviations. The P value for the two groups (indicated with an asterisk) was calculated to be <0.0143 based on a paired t test.

FIG. 11.

Schematic representation of functions associated with the human NatC complex. (A) The hNatC complex, composed of the subunits hMak3, hMak10, and hMak31, associates with ribosomes and acetylates nascent Met-Leu and similar polypeptides. One protein of this type, the Arf-like GTPase hArl8b, depends on the N-terminal acetylation for its localization to lysosomes. (B) Knockdown of hNatC complex subunits by siRNA most likely reduces the N-terminal acetylation of some NatC-type substrates. Directly or indirectly, this stabilizes and activates p53, which in turn induces transcription of genes leading to the induction of apoptosis. Apoptosis decreases the protein level of hMak3, while the mRNA level is unaffected.