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. 2006 Feb;5(2):368-78.
doi: 10.1128/EC.5.2.368-378.2006.

Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB

Robert Caesar  1 , Jonas Warringer, Anders Blomberg
Affiliations

Physiological importance and identification of novel targets for the N-terminal acetyltransferase NatB

Robert Caesar et al. Eukaryot Cell. 2006 Feb.

Abstract

The N-terminal acetyltransferase NatB in Saccharomyces cerevisiae consists of the catalytic subunit Nat3p and the associated subunit Mdm20p. We here extend our present knowledge about the physiological role of NatB by a combined proteomics and phenomics approach. We found that strains deleted for either NAT3 or MDM20 displayed different growth rates and morphologies in specific stress conditions, demonstrating that the two NatB subunits have partly individual functions. Earlier reported phenotypes of the nat3Delta strain have been associated with altered functionality of actin cables. However, we found that point mutants of tropomyosin that suppress the actin cable defect observed in nat3Delta only partially restores wild-type growth and morphology, indicating the existence of functionally important acetylations unrelated to actin cable function. Predicted NatB substrates were dramatically overrepresented in a distinct set of biological processes, mainly related to DNA processing and cell cycle progression. Three of these proteins, Cac2p, Pac10p, and Swc7p, were identified as true NatB substrates. To identify N-terminal acetylations potentially important for protein function, we performed a large-scale comparative phenotypic analysis including nat3Delta and strains deleted for the putative NatB substrates involved in cell cycle regulation and DNA processing. By this procedure we predicted functional importance of the N-terminal acetylation for 31 proteins.

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Figures

FIG. 1.
FIG. 1.
Phenotypes for nat3Δ and mdm20Δ. (A) Growth in basal medium. Filled squares represent wild-type culture, open circles represent mdm20Δ culture, and open squares represent nat3Δ culture. Microscopy pictures are not in the same scale. (B) Logarithmic phenotypic index (LPI) values for growth rate. White bars represent nat3Δ, and black bars represent mdm20Δ. Two stars indicate significance (P < 0.001) as defined in Materials and Methods.
FIG. 2.
FIG. 2.
Portions of 2D-PAGE gels of protein extracts prepared from wild-type, nat3Δ, and mdm20Δ cells. The positions of the acetylated and the unacetylated proteins are indicated by circles in each picture. Due to the loss of the N terminus the acetyl group proteins are moved toward the basic end of the gel in the deletion mutant strains.
FIG. 3.
FIG. 3.
Recording of growth and LSC values for nat3Δ and nat3Δ with a TPM1-5 mutant. (A) Growth in basal medium. Squares represent the wild type and circles represent nat3Δ. Filled symbols represent cells carrying a p[CEN URA3] plasmid, empty symbols represent cells carrying a p[CEN URA3 TPM1-5] plasmid. (B) Microscopy pictures of cells grown in defined medium in liquid media. (C) LSC values for growth rate. Black bars represent nat3Δ, and white bars represent nat3Δ with a TPM1-5 mutant.
FIG. 4.
FIG. 4.
Codon adaptation index (CAI) and overrepresentation in functional and localization categories of proteins with MD and ME as N-terminal residues. (A) Representation of all S. cerevisiae proteins (black bars) and protein with MD and ME as terminal residues (white bars) within given CAI intervals. Significance of enrichment of proteins with MD or ME as terminal residues within categories of (B) cellular functions and (C) localization. Numbers given after category names represent fold enrichments. Overrepresentation corresponds to a P value of 0.001.
FIG. 5.
FIG. 5.
Western blot of isoelectrically focused proteins. Proteins fused with C-terminal TAP tags extracted from wild-type and from nat3Δ cells. Due to the loss of the N-terminal acetyl group, proteins in nat3Δ resolve at a more basic pI. wt, wild type.
FIG. 6.
FIG. 6.
Phenotypic similarity between nat3Δ and strains deleted for putative NatB targets. (A) Hierarchical clustering of nat3Δ and strains deleted for putative NatB targets. The strains were subjected to average linkage hierarchical clustering using an uncentered Pearson correlation coefficient as a similarity measurement. (B) LPI values for growth rate for nat3Δ (black bars) and for strains deleted in genes coding for putative NatB substrates (white bars) exhibiting significant sensitivity to hydroxyurea and ethidium bromide.
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