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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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Protein N-acetylation inhibits ER translocation both in vivo and invitro.(A) Schematic of wild-type and P2 signal sequence mutants of Pdi1p andpreproα-factor. Position of N-glycosylation (ψ) and signalpeptidase cleavage (↓) sites are indicated. (B) Wild-type and indicatedmutants of myc-tagged Pdi1p and ppαF were expressed in wild-type(Δpep4) or sec61-3 strains, andtreated, where indicated, with Tunicamycin (Tu). Steady-state levels ofprotein were determined by preparation of cell extracts from these strainsand analysis by Western blot with anti-myc antibodies. (C) Wild-type (MR)and MS forms of lysine-less ppαF (where all lysines had been mutated toarginine) were translated in vitro, then incubated with yeast microsomes(yRM). Position of non-translocated (ppαF) and signal-sequence cleaved,glycosylated (g-pαF) are indicated. (D) Lysine-less forms of bothwild-type (MR) and MS ppαF were translated in vitro in the presence ofeither [35S] methionine or [14C]acetyl-CoA and immuno-precipitated with anti-ppαF antibodies beforeanalysis by either scintillation counting or SDS-PAGE. Error bars representstandard deviation; three asterisks indicate p<0.001according to the two-tailed student's t test. (E)Wild-type (MR) and MS ppαF with lysine residues at positions 5 and 12were translated in vitro in the presence of [35S]methionine and TDBA-lysyl-tRNA. Targeting to microsomes was performed in theabsence of ATP and then cross-linking induced by uv-irradiation. Whereindicated, samples were denatured and immuno-precipitated with Sec61antisera.
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pbio-1001073-g004: Protein N-acetylation inhibits ER translocation both in vivo and invitro.(A) Schematic of wild-type and P2 signal sequence mutants of Pdi1p andpreproα-factor. Position of N-glycosylation (ψ) and signalpeptidase cleavage (↓) sites are indicated. (B) Wild-type and indicatedmutants of myc-tagged Pdi1p and ppαF were expressed in wild-type(Δpep4) or sec61-3 strains, andtreated, where indicated, with Tunicamycin (Tu). Steady-state levels ofprotein were determined by preparation of cell extracts from these strainsand analysis by Western blot with anti-myc antibodies. (C) Wild-type (MR)and MS forms of lysine-less ppαF (where all lysines had been mutated toarginine) were translated in vitro, then incubated with yeast microsomes(yRM). Position of non-translocated (ppαF) and signal-sequence cleaved,glycosylated (g-pαF) are indicated. (D) Lysine-less forms of bothwild-type (MR) and MS ppαF were translated in vitro in the presence ofeither [35S] methionine or [14C]acetyl-CoA and immuno-precipitated with anti-ppαF antibodies beforeanalysis by either scintillation counting or SDS-PAGE. Error bars representstandard deviation; three asterisks indicate p<0.001according to the two-tailed student's t test. (E)Wild-type (MR) and MS ppαF with lysine residues at positions 5 and 12were translated in vitro in the presence of [35S]methionine and TDBA-lysyl-tRNA. Targeting to microsomes was performed in theabsence of ATP and then cross-linking induced by uv-irradiation. Whereindicated, samples were denatured and immuno-precipitated with Sec61antisera.

Mentions: We next examined the effect of mutants predicted to induce acetylation of twoindependent ER translocation substrates, namely Pdi1p and prepro-alpha factor(ppαF) (Figure 4A and 4B).The signal sequence of Pdi1p begins MK and hence is not predicted to be a substratefor MetAP or N-acetylation [2]. MSK and MEK mutations both led to accumulation ofnon-translocated precursor and a reduction of fully translocated glycosylated Pdi1pat steady state. Furthermore, analysis by mass-spectrometry confirmed that the MSKmutant of pPdi1 was methionine-processed and N-acetylated in vivo, as predicted(FigureS2). No peptides corresponding to an unmodified N-terminus weredetected.


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

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

Protein N-acetylation inhibits ER translocation both in vivo and invitro.(A) Schematic of wild-type and P2 signal sequence mutants of Pdi1p andpreproα-factor. Position of N-glycosylation (ψ) and signalpeptidase cleavage (↓) sites are indicated. (B) Wild-type and indicatedmutants of myc-tagged Pdi1p and ppαF were expressed in wild-type(Δpep4) or sec61-3 strains, andtreated, where indicated, with Tunicamycin (Tu). Steady-state levels ofprotein were determined by preparation of cell extracts from these strainsand analysis by Western blot with anti-myc antibodies. (C) Wild-type (MR)and MS forms of lysine-less ppαF (where all lysines had been mutated toarginine) were translated in vitro, then incubated with yeast microsomes(yRM). Position of non-translocated (ppαF) and signal-sequence cleaved,glycosylated (g-pαF) are indicated. (D) Lysine-less forms of bothwild-type (MR) and MS ppαF were translated in vitro in the presence ofeither [35S] methionine or [14C]acetyl-CoA and immuno-precipitated with anti-ppαF antibodies beforeanalysis by either scintillation counting or SDS-PAGE. Error bars representstandard deviation; three asterisks indicate p<0.001according to the two-tailed student's t test. (E)Wild-type (MR) and MS ppαF with lysine residues at positions 5 and 12were translated in vitro in the presence of [35S]methionine and TDBA-lysyl-tRNA. Targeting to microsomes was performed in theabsence of ATP and then cross-linking induced by uv-irradiation. Whereindicated, samples were denatured and immuno-precipitated with Sec61antisera.
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Related In: Results  -  Collection

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Show All Figures
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pbio-1001073-g004: Protein N-acetylation inhibits ER translocation both in vivo and invitro.(A) Schematic of wild-type and P2 signal sequence mutants of Pdi1p andpreproα-factor. Position of N-glycosylation (ψ) and signalpeptidase cleavage (↓) sites are indicated. (B) Wild-type and indicatedmutants of myc-tagged Pdi1p and ppαF were expressed in wild-type(Δpep4) or sec61-3 strains, andtreated, where indicated, with Tunicamycin (Tu). Steady-state levels ofprotein were determined by preparation of cell extracts from these strainsand analysis by Western blot with anti-myc antibodies. (C) Wild-type (MR)and MS forms of lysine-less ppαF (where all lysines had been mutated toarginine) were translated in vitro, then incubated with yeast microsomes(yRM). Position of non-translocated (ppαF) and signal-sequence cleaved,glycosylated (g-pαF) are indicated. (D) Lysine-less forms of bothwild-type (MR) and MS ppαF were translated in vitro in the presence ofeither [35S] methionine or [14C]acetyl-CoA and immuno-precipitated with anti-ppαF antibodies beforeanalysis by either scintillation counting or SDS-PAGE. Error bars representstandard deviation; three asterisks indicate p<0.001according to the two-tailed student's t test. (E)Wild-type (MR) and MS ppαF with lysine residues at positions 5 and 12were translated in vitro in the presence of [35S]methionine and TDBA-lysyl-tRNA. Targeting to microsomes was performed in theabsence of ATP and then cross-linking induced by uv-irradiation. Whereindicated, samples were denatured and immuno-precipitated with Sec61antisera.
Mentions: We next examined the effect of mutants predicted to induce acetylation of twoindependent ER translocation substrates, namely Pdi1p and prepro-alpha factor(ppαF) (Figure 4A and 4B).The signal sequence of Pdi1p begins MK and hence is not predicted to be a substratefor MetAP or N-acetylation [2]. MSK and MEK mutations both led to accumulation ofnon-translocated precursor and a reduction of fully translocated glycosylated Pdi1pat steady state. Furthermore, analysis by mass-spectrometry confirmed that the MSKmutant of pPdi1 was methionine-processed and N-acetylated in vivo, as predicted(FigureS2). No peptides corresponding to an unmodified N-terminus weredetected.

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