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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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Removal of the N-terminal methionine inhibits ER translocation ofCPY.(A) Schematic of wild-type CPY and P2 mutants. Signal peptide sequence,position of N-glycosylation (ψ), and signal peptidase cleavage (↓)sites are indicated. (B) Yeast cells (Δpep4,Δprc1)expressing either wild-type or mutant CPY were pulse-labelled with[35S]methionine/cysteine, then CPYimmunoprecipitated, and analysed by SDS-PAGE and phosphorimaging. Positionsof glycosylated CPY (g4-pCPY and g3-pCPY) are indicated as are theuntranslocated ppCPY and signal-sequence cleaved, non-glycosylated CPY(pCPY) observed in sec61-3 cells and in tunicamycin-treatedwild-type cells (Tu), respectively. Translocation efficiency was determinedby quantification of ppCPY and g3- and g4-pCPY from three independentexperiments. Error bars represent standard error of the mean. Asterisksrepresent p<0.05 (*) and p<0.001(***) according to the one-way analysis of variance withTukey's multiple comparison test. (C) CPY translocation was analysed asin (B), in a wild-type (Δpep4,Δprc1) and isogenicΔmap1 strain in the presence and absence of theMap2 inhibitor fumagillin (for quantification, see FigureS1).
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pbio-1001073-g002: Removal of the N-terminal methionine inhibits ER translocation ofCPY.(A) Schematic of wild-type CPY and P2 mutants. Signal peptide sequence,position of N-glycosylation (ψ), and signal peptidase cleavage (↓)sites are indicated. (B) Yeast cells (Δpep4,Δprc1)expressing either wild-type or mutant CPY were pulse-labelled with[35S]methionine/cysteine, then CPYimmunoprecipitated, and analysed by SDS-PAGE and phosphorimaging. Positionsof glycosylated CPY (g4-pCPY and g3-pCPY) are indicated as are theuntranslocated ppCPY and signal-sequence cleaved, non-glycosylated CPY(pCPY) observed in sec61-3 cells and in tunicamycin-treatedwild-type cells (Tu), respectively. Translocation efficiency was determinedby quantification of ppCPY and g3- and g4-pCPY from three independentexperiments. Error bars represent standard error of the mean. Asterisksrepresent p<0.05 (*) and p<0.001(***) according to the one-way analysis of variance withTukey's multiple comparison test. (C) CPY translocation was analysed asin (B), in a wild-type (Δpep4,Δprc1) and isogenicΔmap1 strain in the presence and absence of theMap2 inhibitor fumagillin (for quantification, see FigureS1).

Mentions: We next addressed whether this bias was of functional significance for ERtranslocation. The signal sequence of Carboxypeptidase Y (CPY) [38] begins with “MK”and so, like most secretory proteins in our analysis, is predicted to remainunprocessed. Rather than mutating the native P2 residue we chose to insert one ofseven different amino acids between the initiator methionine and the followinglysine residue (Figure 2A). Wethen assessed the translocation efficiency of these mutants in vivo by monitoringtheir ER-dependent glycosylation (Figure 2B). Insertion of arginine or valine had no effect on theefficiency of translocation, demonstrating that an insertion at this position doesnot inherently perturb signal sequence function. However, the other five insertionstested all resulted in translocation defects indicated by the accumulation of thecytosolic precursor form of preproCPY (ppCPY). The most significant defects wereobserved for glycine, serine, and glutamate, which are three of the four residuesmost biased in their frequency distribution towards cytosolic proteins (Figure 1B). Thus the bias observedin our bioinformatic analysis correlates with defects in translocation, therebyimplying an important role for P2 in a functional signal sequence.


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

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

Removal of the N-terminal methionine inhibits ER translocation ofCPY.(A) Schematic of wild-type CPY and P2 mutants. Signal peptide sequence,position of N-glycosylation (ψ), and signal peptidase cleavage (↓)sites are indicated. (B) Yeast cells (Δpep4,Δprc1)expressing either wild-type or mutant CPY were pulse-labelled with[35S]methionine/cysteine, then CPYimmunoprecipitated, and analysed by SDS-PAGE and phosphorimaging. Positionsof glycosylated CPY (g4-pCPY and g3-pCPY) are indicated as are theuntranslocated ppCPY and signal-sequence cleaved, non-glycosylated CPY(pCPY) observed in sec61-3 cells and in tunicamycin-treatedwild-type cells (Tu), respectively. Translocation efficiency was determinedby quantification of ppCPY and g3- and g4-pCPY from three independentexperiments. Error bars represent standard error of the mean. Asterisksrepresent p<0.05 (*) and p<0.001(***) according to the one-way analysis of variance withTukey's multiple comparison test. (C) CPY translocation was analysed asin (B), in a wild-type (Δpep4,Δprc1) and isogenicΔmap1 strain in the presence and absence of theMap2 inhibitor fumagillin (for quantification, see FigureS1).
© Copyright Policy
Related In: Results  -  Collection

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pbio-1001073-g002: Removal of the N-terminal methionine inhibits ER translocation ofCPY.(A) Schematic of wild-type CPY and P2 mutants. Signal peptide sequence,position of N-glycosylation (ψ), and signal peptidase cleavage (↓)sites are indicated. (B) Yeast cells (Δpep4,Δprc1)expressing either wild-type or mutant CPY were pulse-labelled with[35S]methionine/cysteine, then CPYimmunoprecipitated, and analysed by SDS-PAGE and phosphorimaging. Positionsof glycosylated CPY (g4-pCPY and g3-pCPY) are indicated as are theuntranslocated ppCPY and signal-sequence cleaved, non-glycosylated CPY(pCPY) observed in sec61-3 cells and in tunicamycin-treatedwild-type cells (Tu), respectively. Translocation efficiency was determinedby quantification of ppCPY and g3- and g4-pCPY from three independentexperiments. Error bars represent standard error of the mean. Asterisksrepresent p<0.05 (*) and p<0.001(***) according to the one-way analysis of variance withTukey's multiple comparison test. (C) CPY translocation was analysed asin (B), in a wild-type (Δpep4,Δprc1) and isogenicΔmap1 strain in the presence and absence of theMap2 inhibitor fumagillin (for quantification, see FigureS1).
Mentions: We next addressed whether this bias was of functional significance for ERtranslocation. The signal sequence of Carboxypeptidase Y (CPY) [38] begins with “MK”and so, like most secretory proteins in our analysis, is predicted to remainunprocessed. Rather than mutating the native P2 residue we chose to insert one ofseven different amino acids between the initiator methionine and the followinglysine residue (Figure 2A). Wethen assessed the translocation efficiency of these mutants in vivo by monitoringtheir ER-dependent glycosylation (Figure 2B). Insertion of arginine or valine had no effect on theefficiency of translocation, demonstrating that an insertion at this position doesnot inherently perturb signal sequence function. However, the other five insertionstested all resulted in translocation defects indicated by the accumulation of thecytosolic precursor form of preproCPY (ppCPY). The most significant defects wereobserved for glycine, serine, and glutamate, which are three of the four residuesmost biased in their frequency distribution towards cytosolic proteins (Figure 1B). Thus the bias observedin our bioinformatic analysis correlates with defects in translocation, therebyimplying an important role for P2 in a functional signal sequence.

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
Related in: MedlinePlus