Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC

Abstract

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.

Keywords: N-alpha-acetyltransferase 30; N-terminal acetylation; N-terminal acetylome; S. cerevisiae disease model; glycerol metabolism; mitochondrial metabolism; non-fermentable sugar phenotype; protein modification; subcellular fractionation; virus assembly.

Figure 1

Human NAA30 rescues Arl3 localization and Nfs^–phenotypes of naa30Δ yeast.A, live imaged Arl3-GFP yeast of the indicated genotypes. Shown are representative zoomed fields (scale bar represents 2.5 μm), based on images from > 100 yeast cells from approximately 20 random fields of view per genotype. B and C, cells were cultured in medium with either glucose (B) or glycerol (C) as the sole carbon source and the cell density was measured by optical density (A₆₀₀). The bar charts show the increased cell density, A_600, from initial to late exponential growth phase. All data shown represent the mean of three technical replicates from two biological replicates (sister clones) per genotype (n = 6 in total per genotype), and all error bars show SD. Data were analyzed by a two-tailed t test with unequal variance. ∗∗∗ indicate p-value <0.0005, ∗∗∗∗ indicate p-value <0.00005 and other comparisons to WT (+hNAA50 and +hNAA60) were non-significant.

Figure 2

Preparation and validation of samples for N-terminomics analysis.A, yeast extracts were prepared by two methods, classic bead-beating lysis providing a total lysate (a) and yeast subcellular fractionation providing three samples (b-d) enriched for various cellular compartments. P₂₀₀₀/S₂₀₀₀: pellet/supernatant after centrifugation at 2000g; P_100,000/S_100,000: pellet/supernatant after centrifugation at 100,000g. B, Western blot analysis of yeast subcellular fractions prepared for N-terminomics (COFRADIC). The indicated yeast strains WT, naa30Δ, and naa30Δ-[hNAA30] were subjected to subcellular fractionation as described in panel A and Experimental procedures. Aliquots were collected for Western blot inspection and probed using antibodies against the indicated marker proteins. Labels on the right side summarize the final segregation of each marker. C, in further preparative steps, the pellet samples were divided into soluble and insoluble fractions that were analyzed separately, yielding in total 18 proteome fractions (three different yeast strains and six cellular fractions). COFRADIC, combined fractional diagonal chromatography.

Figure 3

Among proteins with an N-terminus of the NatC-type category, proteins with analternative,internal N-terminus are more frequently Nt-acetylated than proteins with a canonical N-terminus. The protein N-termini detected in this dataset were divided into two groups according to their N-terminal start position. Either starting at position 1 or 2 (labeled 1 or 2) or after amino acid position 2 (labeled >2), within each NAT substrate class, the fraction of Nt-acetylated N-termini was calculated. NAT, N-terminal acetyltransferase.

Figure 4

Human NAA30 can (partially) restore yeast NatC substrate Nt-acetylation. Representative MS-spectra of two yeast NatC substrates (A) partially (SSC1 (ML-), P0CCS90) or (B) fully (YDR239C (MF-), Q03780) restored in their Nt-acetylation status by ectopic expression of hNAA30 in naa30Δ yeast. NAT, N-terminal acetyltransferase.

Figure 5

N-terminal substrate specificities of Saccharomyces cerevisiae NATs and their proteome coverage.A, overview of the S. cerevisiae N-terminal acetyltransferases and their preferred N-terminal substrate specificities and proteome coverage. Yeast NatC is known to Nt-acetylate ML-, MI-, and MF- and MW-starting N-termini, and based on the results presented in the current study, also MY-, MK-, and MM- in addition to some MA-, MV- and MS-starting N-termini. NatD/Naa40 specifically Nt-acetylates histones H2A, H2A.Z, and H4 in addition to a few other SG-starting protein and is therefore not shown in the pie chart. Naa50 may associate with the NatA subunits to form NatE. B, fates of three classes of protein N-termini. Additional N-termini can be considered putative NatC substrates based on the types of N-termini revealed by the current N-terminome analysis, not only to cover additional M-«hydrophobic/amphipathic» N-termini within the NatC/E/other substrate class but also some non-MetAP–processed M-«small» type N-termini. C, estimated tendency for the indicated N-terminus types to be targeted by Nt-acetylation. For example, 23/31 (74%) of the here detected MQ-starting N-termini were NAT substrates. The size of the Nt-acetylome as shown in A is estimated by combining the % Nt-acetylation coverage (shown in in C) with the proteome-abundance of the N-terminus type. MetAP, methionine aminopeptidase; NAT, N-terminal acetyltransferase.