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. 2017 Oct 10;12(10):e0186278.
doi: 10.1371/journal.pone.0186278. eCollection 2017.

Probing the interaction between NatA and the ribosome for co-translational protein acetylation

Robert S Magin  1   2 , Sunbin Deng  1   3 , Haibo Zhang  3 , Barry Cooperman  3 , Ronen Marmorstein  1   3
Affiliations

Probing the interaction between NatA and the ribosome for co-translational protein acetylation

Robert S Magin et al. PLoS One. .

Abstract

N-terminal acetylation is among the most abundant protein modifications in eukaryotic cells. Over the last decade, significant progress has been made in elucidating the function of N-terminal acetylation for a number of diverse systems, involved in a wide variety of biological processes. The enzymes responsible for the modification are the N-terminal acetyltransferases (NATs). The NATs are a highly conserved group of enzymes in eukaryotes, which are responsible for acetylating over 80% of the soluble proteome in human cells. Importantly, many of these NATs act co-translationally; they interact with the ribosome near the exit tunnel and acetylate the nascent protein chain as it is being translated. While the structures of many of the NATs have been determined, the molecular basis for the interaction with ribosome is not known. Here, using purified ribosomes and NatA, a very well-studied NAT, we show that NatA forms a stable complex with the ribosome in the absence of other stabilizing factors and through two conserved regions; primarily through an N-terminal domain and an internal basic helix. These regions may orient the active site of the NatA to face the peptide emerging from the exit tunnel. This work provides a framework for understanding how NatA and potentially other NATs interact with the ribosome for co-translational protein acetylation and sets the foundation for future studies to decouple N-terminal acetyltransferase activity from ribosome association.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. NatA binds to the ribosome in vitro.
(A) Fractions of NatA eluting off of a Superose 6 column. The upper band is Naa15 and the lower band is Naa10. (B) Fractions of the ribosome and an excess of NatA eluting off of a Superose 6 column. Note the excess NatA eluting in fraction 12.
Fig 2
Fig 2. The NatA ribosome interaction has low μM affinity and is salt dependent.
(A) Representative Western blot of NatA/ribosome co-sedimentation assay with increasing concentration of ribosome. P stands for pellet, and S stands for supernatant. The western blot targets the His tag on the Naa15 subunit (Naa10 is untagged) (B) An affinity curve of the ribosome NatA interaction quantified from the co-sedimentation assay. Assay was performed in duplicate (C) Co-sedimentation assay in increasing KCl concentrations. The first two lanes are NatA without ribosome present. S stands for supernatant and P stands for pellet. Uncropped gels are shown in S1 Fig.
Fig 3
Fig 3. Conservation analysis and electrostatic surface of NatA show two regions important for ribosome binding.
(A) A cartoon representation of the NatA complex. Naa10 is shown in cyan, Naa15 in green, and the peptide substrate in magenta. The N-terminus and internal basic helix are indicated, as is the active site where N-termini are acetylated. (B) Conservation map of the NatA complex. Magenta areas represent regions of high sequence conservation and cyan areas represent regions of low sequence conservation. (C) Electrostatic potential map of NatA. Blue areas represented regions which are electropositive, and red areas represent regions which are electronegative. Electropositive region 1 (EPR1), and electropositive region 2 (EPR2) are indicated.
Fig 4
Fig 4. Pull down analysis of NatA mutants.
(A) Location of NatA mutants used in this study. Mutated regions are shown in yellow. Lysines mutated in K3E and K6E constructs are shown as sticks, and the N-terminal domain deleted in the ΔN constructs is indicated in yellow. (B) Sedimentation assay with NatA mutants. Naa15 is indicated. Note for the ΔN mutants, Naa15 runs lower than WT Naa15. The faint bands above 80 kD in these lanes is not Naa15, but rather impurities from the ribosome prep. These lanes are marked with asterisks. The Naa10 band is obscured by the ribosomal proteins in the gel.
Fig 5
Fig 5. Pull down analysis of controls.
(A) A surface view of the NatA complex. Mutated lysine residues are indicated in orange and labeled with their mutant name. B) Pull down analysis of the NatA mutants. Asterisks are used as in Fig 4. C) Activity analysis of mutants. SASE is a peptide corresponding to a known NatA substrate, and MLGP is a peptide corresponding to a NatE substrate (See materials and methods for full length peptide sequences). Assays were done in triplicate.
Fig 6
Fig 6. ΔN-K6E does not bind to ribosomes.
(A) Binding profile of K9E and (B) ΔN-K6E. These data could not be fit to a binding curve. Compare to Fig 2B (C) Fractions of the ribosome and an excess of K9E and (D) ΔN-K6E eluting off of a Superose 6 column. Compare to Fig 1B.
Fig 7
Fig 7. NatB has positively charged regions in the same configuration as NatA.
(A) Cartoon representation of the NatA and NatB complex. Naa10 is shown in cyan and Naa15 in green. Naa20 is shown in yellow and Naa25 in orange. (B) Electrostatic surface representation of Naa15 and Naa25 with EPR1 and EPR2 on the Naa15 structure, and putative areas on Naa25 corresponding to these regions indicated with arrows.
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References

    1. Aksnes H, Drazic A, Marie M, Arnesen T. First Things First: Vital Protein Marks by N-Terminal Acetyltransferases. Trends Biochem Sci. 2016;41(9):746–60. doi: 10.1016/j.tibs.2016.07.005 - DOI - PubMed
    1. Arnesen T, Gromyko D, Pendino F, Ryningen A, Varhaug JE, Lillehaug JR. Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N-alpha-acetyltransferase complex. Oncogene. 2006;25(31):4350–60. Epub 2006/03/07. doi: 10.1038/sj.onc.1209469 . - DOI - PubMed
    1. Starheim KK, Gromyko D, Evjenth R, Ryningen A, Varhaug JE, Lillehaug JR, et al. Knockdown of human N alpha-terminal acetyltransferase complex C leads to p53-dependent apoptosis and aberrant human Arl8b localization. Mol Cell Biol. 2009;29(13):3569–81. Epub 2009/04/29. doi: 10.1128/MCB.01909-08 ; PubMed Central PMCID: PMC2698767. - DOI - PMC - PubMed
    1. Pavlou D, Kirmizis A. Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway. Apoptosis: an international journal on programmed cell death. 2016;21(3):298–311. doi: 10.1007/s10495-015-1207-0 - DOI - PMC - PubMed
    1. Schiza V, Molina-Serrano D, Kyriakou D, Hadjiantoniou A, Kirmizis A. N-alpha-terminal acetylation of histone H4 regulates arginine methylation and ribosomal DNA silencing. PLoS Genet. 2013;9(9):e1003805 Epub 2013/09/27. doi: 10.1371/journal.pgen.1003805 ; PubMed Central PMCID: PMC3778019. - DOI - PMC - PubMed

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