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. 2016 Nov;15(11):3361-3372.
doi: 10.1074/mcp.M116.061010. Epub 2016 Sep 30.

A Role for Human N-alpha Acetyltransferase 30 (Naa30) in Maintaining Mitochondrial Integrity

Petra Van Damme  1   2 , Thomas V Kalvik  3 , Kristian K Starheim  3   4   5 , Veronique Jonckheere  6   2 , Line M Myklebust  3 , Gerben Menschaert  7 , Jan Erik Varhaug  4   8 , Kris Gevaert  6   2 , Thomas Arnesen  3   8
Affiliations

A Role for Human N-alpha Acetyltransferase 30 (Naa30) in Maintaining Mitochondrial Integrity

Petra Van Damme et al. Mol Cell Proteomics. 2016 Nov.

Abstract

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.

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Figures

Fig. 1.
Fig. 1.
Experimental workflow and confirmation of the efficiency of hNAA30-knockdown. A, Workflow of sample preparations for N-terminal COFRADIC. B, Immunoblots of A-431 cell lysates treated with non-targeting siRNA (siCTR) or sihNAA30. Cells were harvested 96 h post siRNA transfection. Blots were probed with anti-Naa30 and anti-Naa35 to assess levels of endogenous Naa30 and Naa35. The asterisk indicates nonspecific bands that serve as loading control.
Fig. 2.
Fig. 2.
The effect of Naa30 knockdown on the Nt-acetylation states of individual proteins and the Nt-acetylome. A, Representative MS spectrum of the N terminus originating from the cell differentiation protein RCD1 homolog (1MHSLATAAPVPTTLAQVDR19). The peptide was fully acetylated in the control setup while being only partially acetylated (83%) in the sihNAA30 knockdown sample. The total concentration of the N terminus remained unaffected in the two samples analyzed (ratio of 1). B, Representative MS spectrum of the N terminus of the hydroxysteroid dehydrogenase-like protein 2 (1MLPNTGR7). The peptide was partially acetylated in both control (32%) and knockdown (3%) samples. The total concentration of the N terminus is significantly lowered upon sihNAA30 knockdown (p ≤ 0.01; ratio of 0.5). C, The scatterplot displays the correlation of the determined degrees of Nt-acetylation when comparing the siCTR (X-axis) and the sihNAA30(Y-axis) N-terminome data sets (N termini with start position 1 or 2) of the total lysate samples (n = 1065). The circles falling outside the ±10% boundaries (dashed lines) represent N termini displaying a significant reduction in the degree of Nt-acetylation upon sihNAA30 knockdown (2). The circles are color coded according to their indicated NAT substrate specificity profiles (see also NAT type column in supplemental Table S1). sihNAA30 knockdown causes a decrease in the levels of Nt-Ac as compared with the siCTR setup only for specific N termini in the NAT type categories of “NatC” or “Other” N termini.
Fig. 3.
Fig. 3.
hNAA30 knockdown impacts mitochondrial matrix protein levels, a category of proteins enriched for NatC type substrates. A, Density plots of log2 transformed sihNAA30/siCTR ratios of all unique N termini (1149), (processed) mitochondrial N termini (116) and (processed) mitochondrial matrix protein N termini (54) identified in the total lysate setup. B, Bar chart of the NAT-type substrate specificity distribution of all initiator methionine (iMet) starting Swiss-Prot protein entries (25744), mitochondrial proteins excluding mitochondrial matrix proteins (761) and mitochondrial matrix proteins (304).
Fig. 4.
Fig. 4.
sihNAA30-treated cells display fragmented mitochondria. HeLa (A, B) and CAL-62 (C, D, E) cells depleted for Naa30 were immunostained for the mitochondrial marker COX IV-mouse (A) or COX IV-rabbit (C, E). Micrographs were taken with a confocal and Z-stacks were Z-projected to visualize COX IV. DAPI (blue) was used to visualize the nuclei (A, C) in overlay with COX-IV (red). The confocal micrograph in (E) was taken with a 6.5 zoom and deconvoluted. White bars correspond to 10 μm. The percentages of cells with perinuclear and fragmented mitochondria were calculated for each sample (B, D). Data are mean and standard deviations from three independent experiments (n = 100–150). The difference between siCTR and sihNAA30 treated cells was statistically significant (p < 0.05, student t test). (F) Immunoblots of cell lysates from HeLa cells treated with nontargeting siRNA (siCTR) or sihNAA30. Cells were harvested 72 h post siRNA transfection. Blots were probed with anti-Naa30 to assess levels of endogenous Naa30. β-tubulin was used as loading control.
Fig. 5.
Fig. 5.
MitoTracker Red CMXRos staining reveals non-functional mitochondria in sihNAA30 treated cells. Confocal micrographs of siRNA treated HeLa cells stained with MitoTracker Red CMXRos. All cells displaying a phenotype of fragmented mitochondria are not or only partially stained by MitoTracker Red CMXRos. At least 100 cells in three independent parallels were observed. DAPI was used to visualize nuclei. White bar indicates 10 μm.
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