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N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum.

Forte GM, Pool MR, Stirling CJ - PLoS Biol. (2011)

Bottom Line: Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way.Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery.Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

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N-terminal acetylation blocks protein translocation.Translocation of wild-type, MS, and ME mutants of CPY was examined z(as inFigure 2B) inwild-type and Δard1 and Δnat3strains, which lack NatA and NatB activity, respectively. Data arerepresentative of three independent experiments.
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pbio-1001073-g003: N-terminal acetylation blocks protein translocation.Translocation of wild-type, MS, and ME mutants of CPY was examined z(as inFigure 2B) inwild-type and Δard1 and Δnat3strains, which lack NatA and NatB activity, respectively. Data arerepresentative of three independent experiments.

Mentions: In our analysis, the ME and MS mutations had the strongest effects on translocation(Figure 2B) and these P2residues displayed extreme bias against their occurrence in natural signal sequences(Figure 1B). While“ME” is not a substrate for MetAP, it is known to promoteN-α-acetylation of the N-terminal methionine by NatB [6]. Likewise, the P2 serine, oncerevealed by MetAP, is predicted to be N-α-acetylated by NatA. We thereforetested whether acetylation might be the key determinant affecting translocation byanalysing translocation efficiencies in either NatA(Δard1) orNatB(Δnat3)-deficient strains (Figure 3). In Δard1 cells,translocation of MS-CPY appeared largely restored while the ME mutant remainedunaffected. The converse was observed in the Δnat3 strain.Importantly, the ability of the different Nat mutants to rescueprecursor translocation matched precisely the substrate specificities of NatA andNatB for MS and ME, respectively. Moreover, the observation that inhibition of MAPactivity specifically rescues the translocation of NatA substrates is entirelyconsistent with methionine cleavage being a prerequisite for NatA-dependentacetylation. Thus, it is the N-α-acetylation of these substrates that is themajor determinant in the inhibition of translocation in vivo.


N-terminal acetylation inhibits protein targeting to the endoplasmic reticulum.

Forte GM, Pool MR, Stirling CJ - PLoS Biol. (2011)

N-terminal acetylation blocks protein translocation.Translocation of wild-type, MS, and ME mutants of CPY was examined z(as inFigure 2B) inwild-type and Δard1 and Δnat3strains, which lack NatA and NatB activity, respectively. Data arerepresentative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104963&req=5

pbio-1001073-g003: N-terminal acetylation blocks protein translocation.Translocation of wild-type, MS, and ME mutants of CPY was examined z(as inFigure 2B) inwild-type and Δard1 and Δnat3strains, which lack NatA and NatB activity, respectively. Data arerepresentative of three independent experiments.
Mentions: In our analysis, the ME and MS mutations had the strongest effects on translocation(Figure 2B) and these P2residues displayed extreme bias against their occurrence in natural signal sequences(Figure 1B). While“ME” is not a substrate for MetAP, it is known to promoteN-α-acetylation of the N-terminal methionine by NatB [6]. Likewise, the P2 serine, oncerevealed by MetAP, is predicted to be N-α-acetylated by NatA. We thereforetested whether acetylation might be the key determinant affecting translocation byanalysing translocation efficiencies in either NatA(Δard1) orNatB(Δnat3)-deficient strains (Figure 3). In Δard1 cells,translocation of MS-CPY appeared largely restored while the ME mutant remainedunaffected. The converse was observed in the Δnat3 strain.Importantly, the ability of the different Nat mutants to rescueprecursor translocation matched precisely the substrate specificities of NatA andNatB for MS and ME, respectively. Moreover, the observation that inhibition of MAPactivity specifically rescues the translocation of NatA substrates is entirelyconsistent with methionine cleavage being a prerequisite for NatA-dependentacetylation. Thus, it is the N-α-acetylation of these substrates that is themajor determinant in the inhibition of translocation in vivo.

Bottom Line: Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way.Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery.Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

ABSTRACT
Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%-80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species.

Show MeSH