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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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An SRP-dependent precursor is refractory to N-acetylation.(A) Schematic of wild-type OPY (CPY with the endogenous signal sequencereplaced by that of Ost1) and corresponding P2 signal sequence mutants. (B)Wild-type and mutant OPY translocation in vivo was monitored bypulse-labelling and immunoprecipitation as in Figure 2B. (C) Lysine-less wild-type (MR)and MS opαF (ppαF with the signal sequence replaced with that ofOst1p and all lysines mutated to arginine) were translated in vitro in thepresence of [35S] methionine, denatured, and modifiedwith amine-reactive sulfo-NHS-SS-biotin. Biotinylated proteins werere-isolated on immobilized-streptavidin and analysed by SDS-PAGE andphosphorimaging.
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pbio-1001073-g005: An SRP-dependent precursor is refractory to N-acetylation.(A) Schematic of wild-type OPY (CPY with the endogenous signal sequencereplaced by that of Ost1) and corresponding P2 signal sequence mutants. (B)Wild-type and mutant OPY translocation in vivo was monitored bypulse-labelling and immunoprecipitation as in Figure 2B. (C) Lysine-less wild-type (MR)and MS opαF (ppαF with the signal sequence replaced with that ofOst1p and all lysines mutated to arginine) were translated in vitro in thepresence of [35S] methionine, denatured, and modifiedwith amine-reactive sulfo-NHS-SS-biotin. Biotinylated proteins werere-isolated on immobilized-streptavidin and analysed by SDS-PAGE andphosphorimaging.

Mentions: There are two pathways by which secretory precursors can be targeted to the ER; someprecursors follow a post-translational Sec62p-dependent pathway, while substrateswith more hydrophobic signal sequences utilise a co-translational SRP-dependentmechanism [25],[30]. As CPY, Pdi1p, and ppαF are all translocatedpost-translationally, we therefore sought to compare the behaviour of anSRP-dependent substrate. We chose the well-characterised SRP-dependent substrateOPY, a variant of CPY in which the endogenous signal sequence is replaced with thatof Ost1p [41]. TheOPY signal sequence begins MR, and so should remain unprocessed, enabling us toperform a precisely parallel mutational analysis to that for CPY (see Figure 2C). In striking contrastto CPY, we found that the introduction of various processable residues at P2 had noeffect on the translocation of OPY (Figure 5A and 5B). Thus the observed inhibitory effect of an MS mutationon translocation can be suppressed in the context of an SRP-dependent signalsequence. This property was not limited to the Ost1p signal sequence;co-translational translocation of the SRP-dependent substrate DHC-αF[30],[42] into yeastmicrosomes using a yeast translation extract was also unaffected by theincorporation of a potentially acetylatable serine residue at P2 (Figure S4).Moreover, the well-characterized SRP-dependent substrates Sec71 and Dap2 (DPAP B)[30],[43] have P2residues of S and E, respectively, entirely consistent with our finding that NATsubstrates can be tolerated by the SRP pathway.


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

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

An SRP-dependent precursor is refractory to N-acetylation.(A) Schematic of wild-type OPY (CPY with the endogenous signal sequencereplaced by that of Ost1) and corresponding P2 signal sequence mutants. (B)Wild-type and mutant OPY translocation in vivo was monitored bypulse-labelling and immunoprecipitation as in Figure 2B. (C) Lysine-less wild-type (MR)and MS opαF (ppαF with the signal sequence replaced with that ofOst1p and all lysines mutated to arginine) were translated in vitro in thepresence of [35S] methionine, denatured, and modifiedwith amine-reactive sulfo-NHS-SS-biotin. Biotinylated proteins werere-isolated on immobilized-streptavidin and analysed by SDS-PAGE andphosphorimaging.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-1001073-g005: An SRP-dependent precursor is refractory to N-acetylation.(A) Schematic of wild-type OPY (CPY with the endogenous signal sequencereplaced by that of Ost1) and corresponding P2 signal sequence mutants. (B)Wild-type and mutant OPY translocation in vivo was monitored bypulse-labelling and immunoprecipitation as in Figure 2B. (C) Lysine-less wild-type (MR)and MS opαF (ppαF with the signal sequence replaced with that ofOst1p and all lysines mutated to arginine) were translated in vitro in thepresence of [35S] methionine, denatured, and modifiedwith amine-reactive sulfo-NHS-SS-biotin. Biotinylated proteins werere-isolated on immobilized-streptavidin and analysed by SDS-PAGE andphosphorimaging.
Mentions: There are two pathways by which secretory precursors can be targeted to the ER; someprecursors follow a post-translational Sec62p-dependent pathway, while substrateswith more hydrophobic signal sequences utilise a co-translational SRP-dependentmechanism [25],[30]. As CPY, Pdi1p, and ppαF are all translocatedpost-translationally, we therefore sought to compare the behaviour of anSRP-dependent substrate. We chose the well-characterised SRP-dependent substrateOPY, a variant of CPY in which the endogenous signal sequence is replaced with thatof Ost1p [41]. TheOPY signal sequence begins MR, and so should remain unprocessed, enabling us toperform a precisely parallel mutational analysis to that for CPY (see Figure 2C). In striking contrastto CPY, we found that the introduction of various processable residues at P2 had noeffect on the translocation of OPY (Figure 5A and 5B). Thus the observed inhibitory effect of an MS mutationon translocation can be suppressed in the context of an SRP-dependent signalsequence. This property was not limited to the Ost1p signal sequence;co-translational translocation of the SRP-dependent substrate DHC-αF[30],[42] into yeastmicrosomes using a yeast translation extract was also unaffected by theincorporation of a potentially acetylatable serine residue at P2 (Figure S4).Moreover, the well-characterized SRP-dependent substrates Sec71 and Dap2 (DPAP B)[30],[43] have P2residues of S and E, respectively, entirely consistent with our finding that NATsubstrates can be tolerated by the SRP pathway.

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