The Action of N-terminal Acetyltransferases on Yeast Ribosomal Proteins

Randy J Arnold,Bogdan Polevoda,James P Reilly,Fred Sherman

doi:10.1074/jbc.274.52.37035

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to determine the state of N-terminal acetylation of 68 ribosomal proteins from a normal strain of Saccharomyces cerevisiae and from the ard1-Delta, nat3-Delta, and mak3-Delta mutants (), each lacking a catalytic subunit of three different N-terminal acetyltransferases. A total 30 of the of 68 ribosomal proteins were N-terminal-acetylated, and 24 of these (80%) were NatA substrates, unacetylated in solely the ard1-Delta mutant and having mainly Ac-Ser- termini and a few with Ac-Ala- or Ac-Thr- termini. Only 4 (13%) were NatB substrates, unacetylated in solely the nat3-Delta mutant, and having Ac-Met-Asp- or Ac-Met-Glu- termini. No NatC substrates were uncovered, e.g. unacetylated in solely mak3-Delta mutants, consistent with finding that none of the ribosomal proteins had Ac-Met-Ile-, Ac-Met-Leu-, or Ac-Met-Phe- termini. Interestingly, two new types of the unusual NatD substrates were uncovered, having either Ac-Ser-Asp-Phe- or Ac-Ser-Asp-Ala- termini that were unacetylated in the ard1-Delta mutant, and only partially acetylated in the mak3-Delta mutant and, for one case, also only partially in the nat3-Delta mutant. We suggest that the acetylation of NatD substrates requires not only Ard1p and Nat1p, but also auxiliary factors that are acetylated by the Mak3p and Nat3p N-terminal acetyltransferases.

Highlights

Cleavage of N-terminal1 methionine residues and N-terminal acetylation are the most common modifications, occurring cotranslationally on the vast majority of eukaryotic proteins
N-terminal Acetyltransferases in Yeast study with ribosomal proteins has extended the known kinds of NatA and NatB substrates and, more importantly, revealed new types of NatD substrates, which are dependent on all three NATs, Ard1p, Nat3p, and Mak3p
Normal Ribosomal Proteins—Ribosomal proteins from the normal strain and from each of the ard1-⌬, nat3-⌬, and mak3-⌬ mutants were subjected to mass spectrometric analysis

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains—The following strains used in this study have been described by Polevoda et al [4]: B-11679 (MAT␣ ura3–52) (denoted “normal”); B-11706 (ard1-⌬::kanMX2 MAT␣ ura3-52) (denoted ard1-⌬); B-11852 (nat3-⌬::kanMX2 MAT␣ ura3-52) (denoted nat3-⌬); and B-11705 (mak3-⌬::kanMX2MAT␣ ura3-52) (denoted mak3-⌬). The cell extracts were centrifuged three consecutive times at 14,000 ϫ g, the final supernatants layered over a 10% sucrose (w/v) cushion of TMN buffer containing 0.5 M NH4Cl to strip off loosely associated proteins and centrifuged at 50,000 rpm for 150 min in SW 55 rotor (Beckman). Mass Spectrometric Analysis—MALDI samples were prepared by mixing one part ribosome solution with three parts sinapinic acid matrix at a concentration of 10 mg/ml in 2:1 0.1% trifluoroacetic acid/ acetonitrile and applying 1 ␮l of this mixture to the sample probe. 1% trifluoroacetic acid was added to the ribosome sample before mixing with matrix to enhance the MALDI signal. External mass calibration was performed using a sample containing trypsinogen and carbonic anhydrase in sinapinic acid matrix. After identifying some subunit proteins, their measured flight times and masses calculated from genomic data were used to internally calibrate spectra

RESULTS AND DISCUSSION

Protein name

NatD NatD NatA NatA