N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

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.


Introduction
The mechanism of translational initiation dictates that eukaryotic proteins are synthesized with an amino-terminal methionine residue.In 80% of yeast proteins studied, the initiating methionine is removed to reveal a new amino-terminal residue [1], and some 50% of proteins have their amino-terminal residue acetylated [2,3].Hence rather few proteins possess an unmodified Nterminus.However, while N-terminal processing is widespread, its biological significance is not well understood.It has been suggested to contribute to differential protein stability and has recently been shown to function as a degron for certain cytosolic proteins [4,5], while in a small number of cases the processed N-terminus is known to contribute directly to protein function [6][7][8][9].exposed by MetAP cleavage, whereas NatB acetylates methionine residues that are followed by either D, E, or N at P2 [3,16,17].NatC acetylates certain methionines with either L, I, W, or F at P2, but other sequence elements influence processing in this case [18].NatD appears to be specialised for histone N-acetylation [19] and finally NatE acetylates substrates with Leucine at P2 and Proline at P4 [20].
While most proteins remain in the cytoplasm after synthesis, others are targeted to different compartments.Those destined for the secretory pathway typically possess an N-terminal signalsequence which directs them to the endoplasmic reticulum (ER) [21].These proteins are translocated into the lumen of the ER, via the Sec61 translocon, whereupon their signal-sequence is removed by signal peptidase [22].A subset of membrane proteins can be [27][28][29].The alternative ''post-translational'' pathway is independent of SRP/SR [30] and targets full-length polypeptides in a reaction that requires cytosolic chaperones that maintain precur-evident in signal sequences.A clear pattern emerged whe ratio of frequencies were compared between the two clas proteins (Figure 1B); small and acidic residues were strongly b towards cytosolic proteins, whereas large and basic ones favoured in signal sequences.The frequency of small resid P2 in cytosolic proteins predicts that ,72% of these pr would be substrates for MetAP cleavage (Figure 1C), in agreement with empirical data from proteomic studies [2 contrast only 23% of signal sequences would be predicted MetAP substrates (Figure 1C).Hence our data reveal th signal sequences there appears to be a strong selection f residues that would maintain the original N-terminal methio We next addressed whether this bias was of func significance for ER translocation.The signal sequenc Carboxypeptidase Y (CPY) [38] begins with ''MK'' and so most secretory proteins in our analysis, is predicted to re unprocessed.Rather than mutating the native P2 residue we to insert one of seven different amino acids between the in methionine and the following lysine residue (Figure 2A).We assessed the translocation efficiency of these mutants in vi monitoring their ER-dependent glycosylation (Figure 2B).tion of arginine or valine had no effect on the efficien translocation, demonstrating that an insertion at this position not inherently perturb signal sequence function.Howeve other five insertions tested all resulted in translocation d indicated by the accumulation of the cytosolic precursor fo

Author Summary
The eukaryotic cell comprises several distinct compartments, called organelles, required to perform specific functions.The proteins in these compartments are almost always synthesised in the cytoplasm and so require complex sorting mechanisms to ensure their delivery to the appropriate organelle.Of course, not all proteins need to leave the cytoplasm since many remain there to perform cytoplasmic functions.It is well known that many proteins are modified by acetylation of their aminoterminus at a very early stage in their synthesis.We have discovered a profound difference between the likelihood of such a modification on cytoplasmic proteins and on those destined for one of the major organelles, the endoplasmic reticulum (ER): whereas cytoplasmic proteins are typically acetylated, those bound for the ER are largely unmodified.Moreover, when specific ER proteins were engineered to induce their acetylation we found that their targeting to the ER was inhibited.Our data suggest that Nterminal acetylation is a major determinant in protein sorting in eukaryotes.

N-Terminal Acetylation Inhibits ER Translo
Figure 1.Amino acid frequency at P2 of signal sequences versus cytosolic proteins.(A) Relative frequency of amino acids at P2 of a filtered set of 277 signal sequence-containing proteins from S. cerevisiae was compared to a similar size group (n = 252) of randomly selected cytosolic proteins.Frequency distribution between the groups differed significantly (p,0.0001,x 2 = 207.318 df).(B) Ratio of relative frequency of P2 residues between signal sequence (f ss ) and cytosolic (f cyt ) proteins.Tryptophan was absent from the cytosolic group; therefore, no log(f ss /f cyt ) value is plotted.P2 specificities of MetAP, NatA, and NatB are indicated.(C) Predicted methionine cleavage of signal sequence and cytosolic N-termini based on relative P2 frequency.For complete datasets, see Tables S1-S4.doi:10.1371/journal.pbio.1001073.g001among this minority was significantly greater than for the majority subset of sequences (Figure S5).Overall, more than 99% of signal consensus sequence that favours a G at the +4 position (corresponding to the first base of codon 2) in genes optimised be reversed by blocking N-terminal processing, confirming that it is the processing itself that leads to the block in translocation.The bias against N-terminal processing is not restricted to yeast but is also observed across eukaryotes, suggesting this is a widely conserved phenomenon.

N-Terminal Acetylation Inhibits ER Translocation
It is possible that other factors distinct from N-terminal processing might affect the observed bias in amino acid frequency at position 2. We considered the potential effect of the Kozak residue of signal sequences, the strong correlation and clear functional effects make a bias against N-terminal processing the simplest and most likely explanation of the relative P2 residue frequency.
A trivial explanation for the inhibitory effect of acetylation could be the change in charge distribution across the signal sequence, which is known to be important for targeting [39].However, this appears unlikely, firstly as insertion of an additional positively   ORFs (as defined by SGD), and proteins known to be localized to mitochondria, to yield a final filtered set of 277 ORFs.For a complete list of ORFs, see Table S1.The P2 amino acid frequency distribution did not differ significantly between the filtered and unfiltered sets (x 2 = 5.17, 19 df).Graphical and statistical analysis from SGD of proteins with known cytosolic localization.Prediction of N-acetylation was performed as described previously [2]; where appropriate, the P3 residue was also taken into consideration.MN, which is only predicted to lead to Nacetylation in 55% of cases [2], was scored as acetylated.Human ORFs (as defined by SGD), and proteins known to be localized to mitochondria, to yield a final filtered set of 277 ORFs.For a complete list of ORFs, see Table S1.The P2 amino acid frequency distribution did not differ significantly between the filtered and unfiltered sets (x 2 = 5.17, 19 df).Graphical and statistical analysis was performed using Prism 4.0 (GraphPad).MetAP cleavage was assumed for P2 residues A, C, G, P, S, V, and T [11,12].The yeast cytosolic dataset (Table S2) was generated by random selection from SGD of proteins with known cytosolic localization.Prediction of N-acetylation was performed as described previously [2]; where appropriate, the P3 residue was also taken into consideration.MN, which is only predicted to lead to Nacetylation in 55% of cases [2], was scored as acetylated.Human and Caenorhabditis elegans signal sequence datasets were also obtained from the signal peptide database (SPdb) v5. 1 [56].Drosophila melanogaster and Arabidopsis thaliana datasets were obtained Yeast Strains pOPY and pOPY-S, respectively.The PCR products were digested with EcoRI/HincII and cloned into pGF23 (Table S9) to replace the ppaF signal sequence with that of Ost1 or the serine mutant version, respectively.PDI1 was amplified from genomic DNA with appropriate primers (Table S8) that introduce a single

ption and Translation
transcription of various ppaF mRNAs were R from plasmids pEH3 or pGF22 using s (Table S8) and transcription carried out with CPY or anti-aF [60,61]) added to the supernatant.After 1 h, immune complexes were recovered with Protein A sepharose for a further hour and then washed extensively prior to elution with SDS-PAGE sample buffer.Samples were then analysed by SDS-PAGE and visualised either by phosphorimaging or autoradiography.Quantification was performed with Aida image-analyzer software (Raytek).Subsequent statistical analysis was performed using Prism 4.0 (GraphPad).Samples for scintillation counting were dissociated from the sepharose with 3% SDS for 5 min at 95uC.Dissociated protein was dried onto Whatman glass GF/A filter discs and placed in 4.5 mL of scintillant and counted in a Tricarb 2100TR liquid scintillation counter (Packard).

In Vitro Transcription and Translation
Templates for transcription of various ppaF mRNAs were generated by PCR from plasmids pEH3 or pGF22 using appropriate primers (Table S8) and transcription carried out with SP6 polymerase.Transcriptions of OpaF mRNAs were from pGF24 or pGF25 for MR and MS OpaF, respectively, and were carried out with T7 polymerase.Translations were performed in rabbit reticulocyte lysate system (Promega) for 30 min with the inclusion of either 2.04 mCi [ 35 S] Methionine or 0.04 mCi 1-[ 14 C]-Acetyl Coenzyme A (Perkin Elmer) per 10 mL of reaction.Translation was terminated by addition of 2 mM cycloheximide.
Co-translational translocation of D HC -aF into yeast microsomes was performed using translation extracts from a strain overexpressing SRP, as described previously [42].

Yeast Microsomes and Translocation Assays
Preparation of yeast microsomes from a Dpep4 strain was carried out as previously described [62].For translocation assays; 10 mL of translation reaction was incubated with 2 mL microsomes for 20 min at 30uC.

Photocross-Linking
Wild-type and MS K5K14ppaF were translated in rabbit reticulocyte lysate as above but in the presence of e-4-(3-trifluoromethyldiazirino) benzoic acid (TDBA)-lysyl-tRNA and then used for photocross-linking assays as described [63].Briefly, translations were terminated with 2 mM puromycin for 10 min at 30uC, and then treated with 0.5 mg/mL RNase A for 5 min on ice prior to depletion of ATP from the translation reaction and yeast microsomes by treatment with hexokinase/glucose.The microsomes and translation reaction were then combined, allowing targeting to occur for 15 min at 30uC.Microsomes were re-isolated by centrifugation and resuspended in membrane storage buffer.Samples were irradiated added to the supernatant.After 1 h, ecovered with Protein A sepharose for a ashed extensively prior to elution with .Samples were then analysed by SDSer by phosphorimaging or autoradiogs performed with Aida image-analyzer uent statistical analysis was performed ad).Samples for scintillation counting sepharose with 3% SDS for 5 min at was dried onto Whatman glass GF/A 4.5 mL of scintillant and counted in a intillation counter (Packard).

Supporting Information
nd Translation ption of various ppaF mRNAs were plasmids pEH3 or pGF22 using e S8) and transcription carried out with iptions of OpaF mRNAs were from and MS OpaF, respectively, and were erase.Translations were performed in system (Promega) for 30 min with the i [ 35 S] Methionine or 0.04 mCi 1-[ 14 C]rkin Elmer) per 10 mL of reaction.d by addition of 2 mM cycloheximide.cation of D HC -aF into yeast microsomes nslation extracts from a strain overed previously [42].

Figure 2 .
Figure 2. Removal of the N-terminal methionine inhibits ER translocation of CPY.(A) Schematic of wild-type CPY and P2 mutants.Signal peptide sequence, position of N-glycosylation (y), and signal peptidase cleavage (Q) sites are indicated.(B) Yeast cells (Dpep4,Dprc1) expressing either wild-type or mutant CPY were pulse-labelled with [ 35 S]methionine/cysteine, then CPY immunoprecipitated, and analysed by SDS-PAGE and phosphorimaging.Positions of glycosylated CPY (g4-pCPY and g3-pCPY) are indicated as are the untranslocated ppCPY and signal-sequence cleaved, non-glycosylated CPY (pCPY) observed in sec61-3 cells and in tunicamycin-treated wild-type cells (Tu), respectively.Translocation efficiency was determined by quantification of ppCPY and g3-and g4-pCPY from three independent experiments.Error bars represent standard error of the mean.Asterisks represent p,0.05 (*) and p,0.001 (***) according to the one-way analysis of variance with Tukey's multiple comparison test.(C) CPY translocation was analysed as in (B), in a wild-type (Dpep4,Dprc1) and isogenic Dmap1 strain in the presence and absence of the Map2 inhibitor fumagillin (for quantification, see Figure S1).doi:10.1371/journal.pbio.1001073.g002

Figure 2 .
Figure 2. Removal of the N-terminal methionine inhibits ER translocation of CPY.(A) Schematic of wild-type CPY and P2 mutants.Signal peptide sequence, position of N-glycosylation (y), and signal peptidase cleavage (Q) sites are indicated.(B) Yeast cells (Dpep4,Dprc1) expressing either wild-type or mutant CPY were pulse-labelled with [ 35 S]methionine/cysteine, then CPY immunoprecipitated, and analysed by SDS-PAGE and phosphorimaging.Positions of glycosylated CPY (g4-pCPY and g3-pCPY) are indicated as are the untranslocated ppCPY and signal-sequence cleaved, non-glycosylated CPY (pCPY) observed in sec61-3 cells and in tunicamycin-treated wild-type cells (Tu), respectively.Translocation efficiency was determined by quantification of ppCPY and g3-and g4-pCPY from three independent experiments.Error bars represent standard error of the mean.Asterisks represent p,0.05 (*) and p,0.001 (***) according to the one-way analysis of variance with Tukey's multiple comparison test.(C) CPY translocation was analysed as in (B), in a wild-type (Dpep4,Dprc1) and isogenic Dmap1 strain in the presence and absence of the Map2 inhibitor fumagillin (for quantification, see Figure S1).doi:10.1371/journal.pbio.1001073.g002

Figure 3 .
Figure 3. N-terminal acetylation blocks protein translocation.Translocation of wild-type, MS, and ME mutants of CPY was examined z(as in Figure 2B) in wild-type and Dard1 and Dnat3 strains, which lack NatA and NatB activity, respectively.Data are representative of three independent experiments.doi:10.1371/journal.pbio.1001073.g003

Figure 4 .
Figure 4. Protein N-acetylation inhibits ER translocation both in vivo and in vitro.(A) Schematic of wild-type and P2 signal sequence mutants of Pdi1p and preproa-factor.Position of N-glycosylation (y) and signal peptidase cleavage (Q) sites are indicated.(B) Wild-type and indicated mutants of myc-tagged Pdi1p and ppaF were expressed in wild-type (Dpep4) or sec61-3 strains, and treated, where indicated, with Tunicamycin (Tu).Steady-state levels of protein were determined by preparation of cell extracts from these strains and analysis by Western blot with anti-myc antibodies.(C) Wild-type (MR) and MS forms of lysine-less ppaF (where all lysines had been mutated to arginine) were translated in vitro, then incubated with yeast microsomes (yRM).Position of non-translocated (ppaF) and signal-sequence cleaved, glycosylated (g-paF) are indicated.(D) Lysine-less forms of both wild-type (MR) and MS ppaF were translated in vitro in the presence of either [ 35 S] methionine or [ 14 C] acetyl-CoA and immuno-precipitated with anti-ppaF antibodies before analysis by either scintillation counting or SDS-PAGE.Error bars represent standard deviation; three asterisks indicate p,0.001 according to the two-tailed student's t test.(E) Wild-type (MR) and MS ppaF with lysine residues at positions 5 and 12 were translated in vitro in the presence of [ 35 S] methionine and TDBA-lysyl-tRNA.Targeting to microsomes was performed in the absence of ATP and then cross-linking induced by uv-irradiation.Where indicated, samples were denatured and immuno-precipitated with Sec61 antisera.doi:10.1371/journal.pbio.1001073.g004

Figure 4 .
Figure 4. Protein N-acetylation inhibits ER translocation both in vivo and in vitro.(A) Schematic of wild-type and P2 signal sequence mutants of Pdi1p and preproa-factor.Position of N-glycosylation (y) and signal peptidase cleavage (Q) sites are indicated.(B) Wild-type and indicated mutants of myc-tagged Pdi1p and ppaF were expressed in wild-type (Dpep4) or sec61-3 strains, and treated, where indicated, with Tunicamycin (Tu).Steady-state levels of protein were determined by preparation of cell extracts from these strains and analysis by Western blot with anti-myc antibodies.(C) Wild-type (MR) and MS forms of lysine-less ppaF (where all lysines had been mutated to arginine) were translated in vitro, then incubated with yeast microsomes (yRM).Position of non-translocated (ppaF) and signal-sequence cleaved, glycosylated (g-paF) are indicated.(D) Lysine-less forms of both wild-type (MR) and MS ppaF were translated in vitro in the presence of either [ 35 S] methionine or [ 14 C] acetyl-CoA and immuno-precipitated with anti-ppaF antibodies before analysis by either scintillation counting or SDS-PAGE.Error bars represent standard deviation; three asterisks indicate p,0.001 according to the two-tailed student's t test.(E) Wild-type (MR) and MS ppaF with lysine residues at positions 5 and 12 were translated in vitro in the presence of [ 35 S] methionine and TDBA-lysyl-tRNA.Targeting to microsomes was performed in the absence of ATP and then cross-linking induced by uv-irradiation.Where indicated, samples were denatured and immuno-precipitated with Sec61 antisera.doi:10.1371/journal.pbio.1001073.g004

Figure 5 .
Figure 5.An SRP-dependent precursor is refractory to N-acetylation.(A) Schematic of wild-type OPY (CPY with the endogenous signal sequence replaced by that of Ost1) and corresponding P2 signal sequence mutants.(B) Wild-type and mutant OPY translocation in vivo was monitored by pulse-labelling and immunoprecipitation as in Figure 2B.(C) Lysine-less wild-type (MR) and MS opaF (ppaF with the signal sequence replaced with that of Ost1p and all lysines mutated to arginine) were translated in vitro in the presence of [ 35 S] methionine, denatured, and modified with amine-reactive sulfo-NHS-SS-biotin.Biotinylated proteins were re-isolated on immobilized-streptavidin and analysed by SDS-PAGE and phosphorimaging.doi:10.1371/journal.pbio.1001073.g005

Figure 5 .
Figure 5.An SRP-dependent precursor is refractory to N-acetylation.(A) Schematic of wild-type OPY (CPY with the endogenous signal sequence replaced by that of Ost1) and corresponding P2 signal sequence mutants.(B) Wild-type and mutant OPY translocation in vivo was monitored by pulse-labelling and immunoprecipitation as in Figure 2B.(C) Lysine-less wild-type (MR) and MS opaF (ppaF with the signal sequence replaced with that of Ost1p and all lysines mutated to arginine) were translated in vitro in the presence of [ 35 S] methionine, denatured, and modified with amine-reactive sulfo-NHS-SS-biotin.Biotinylated proteins were re-isolated on immobilized-streptavidin and analysed by SDS-PAGE and phosphorimaging.doi:10.1371/journal.pbio.1001073.g005

Figure S1
Figure S1 Quantification of CPY translocation in the presence and absence of MetAP activity.Pulse-labelling of WT (MK) CPY and mutants with A, C, E, G, and S inserted at P2 was performed in wild-type (MAP1 Dprc1 Dpep4) and Dmap1 (Dprc1 Dpep4) yeast cells in the presence and absence of the Map2 inhibitor fumagillin.CPY was immunoprecipitated and analysed by SDS-PAGE and phosphorimaging (see Figure 2).Translocation efficiency was determined from quantification of the relative amounts of glycosylated-CPY and non-translocated pCPY.The data are displayed graphically and represent the means of three independent experiments.Error bars represent the standard error of the mean.Asterisks represent statistically significant differences to the untreated wild-type (MAP1) strain with p,0.01 (**) and p,0.001 (***) according to the two-way analysis of variance.(TIF) Figure S2 MS-pPdi1p is Methionine-cleaved and N-acetylated in

Figure S1
Figure S1 Quantification of CPY translocation in the presence and absence of MetAP activity.Pulse-labelling of WT (MK) CPY and mutants with A, C, E, G, and S inserted at P2 was performed in wild-type (MAP1 Dprc1 Dpep4) and Dmap1 (Dprc1 Dpep4) yeast cells in the presence and absence of the Map2 inhibitor fumagillin.CPY was immunoprecipitated and analysed by SDS-PAGE and phosphorimaging (see Figure 2).Translocation efficiency was determined from quantification of the relative amounts of glycosylated-CPY and non-translocated pCPY.The data are displayed graphically and represent the means of three independent experiments.Error bars represent the standard error of the mean.Asterisks represent statistically significant differences to the untreated wild-type (MAP1) strain with p,0.01 (**) and p,0.001 (***) according to the two-way analysis of variance.(TIF) Figure S2 MS-pPdi1p is Methionine-cleaved and N-acetylated in vivo.MS-pPdi1p-myc was affinity purified from yeast cells with anti-myc antiserum and analysed by SDS-PAGE and staining with Coomassie brilliant blue (Text S1).The MS-pPdi1p-myc precursor band was excised, digested with elastase, and analysed by LC-MS/ MS (Text S1).Product ion spectra and associated fragmentation tables, which list all the fragment ions observed (highlighted), are shown for two N-terminal peptides.No peptides corresponding to an unmodified N-terminus were detected in the analysis.(TIF) Figure S3 N-acetylation of ppaF blocks translocation in vitro.Wild-type (MR), MSR, and MSRR ppaF were translated in vitro in rabbit reticulocyte lysate and then incubated with yeast microsomes (yRM).Position of non-translocated (ppaF) and signal-sequence cleaved, glycosylated (g-paF) are indicated.(*) Ubiquitinylated ppaF generated in the absence of microsomes.(TIF) Figure S4 D HC -aF translocation is insensitive to a P2 residue that can promote N-acetylation.(A) D HC -aF comprises ppaF with the hydrophobic core of the signal sequence replaced with that of DPAP B, creating an SRP-dependent substrate.D HC -aF with the endogenous P2 residue (MR) or with a serine inserted at position 2 (MS) were translated in vitro in a yeast extract supplemented with [ 35 S] methionine in the presence or absence of yeast microsomes (yRM).Translated proteins were immunoprecipitated with anti-aF antibodies prior to analysis by SDS-PAGE and phosphorimaging.Positions of the unprocessed (D HC aF) and glycosylated (g-D HC aF) forms of the protein are indicated.(B) WT and MS ppaF were translated in yeast extract in the presence of [ 35 S] methionine and

Figure S1
Figure S1 Quantification of CPY translocation in the presence and absence of MetAP activity.Pulse-labelling of WT (MK) CPY and mutants with A, C, E, G, and S inserted at P2 was performed in wild-type (MAP1 Dprc1 Dpep4) and Dmap1 (Dprc1 Dpep4) yeast cells in the presence and absence of the Map2 inhibitor fumagillin.CPY was immunoprecipitated and analysed by SDS-PAGE and phosphorimaging (see Figure 2).Translocation efficiency was determined from quantification of the relative amounts of glycosylated-CPY and non-translocated pCPY.The data are displayed graphically and represent the means of three independent experiments.Error bars represent the standard error of the mean.Asterisks represent statistically significant differences to the untreated wild-type (MAP1) strain with p,0.01 (**) and p,0.001 (***) according to the two-way analysis of variance.(TIF) Figure S2 MS-pPdi1p is Methionine-cleaved and N-acetylated in vivo.MS-pPdi1p-myc was affinity purified from yeast cells with anti-myc antiserum and analysed by SDS-PAGE and staining with Coomassie brilliant blue (Text S1).The MS-pPdi1p-myc precursor band was excised, digested with elastase, and analysed by LC-MS/ MS (Text S1).Product ion spectra and associated fragmentation tables, which list all the fragment ions observed (highlighted), are shown for two N-terminal peptides.No peptides corresponding to an unmodified N-terminus were detected in the analysis.(TIF) Figure S3 N-acetylation of ppaF blocks translocation in vitro.Wild-type (MR), MSR, and MSRR ppaF were translated in vitro in rabbit reticulocyte lysate and then incubated with yeast microsomes (yRM).Position of non-translocated (ppaF) and signal-sequence cleaved, glycosylated (g-paF) are indicated.(*) Ubiquitinylated ppaF generated in the absence of microsomes.(TIF) Figure S4 D HC -aF translocation is insensitive to a P2 residue

n
Figure S3 N-acetylation of ppaF blocks translocation in vitro.Wild-type (MR), MSR, and MSRR ppaF were translated in vitro in rabbit reticulocyte lysate and then incubated with yeast microsomes (yRM).Position of non-translocated (ppaF) and signal-sequence cleaved, glycosylated (g-paF) are indicated.(*) Ubiquitinylated ppaF generated in the absence of microsomes.(TIF) Figure S4 D HC -aF translocation is insensitive to a P2 residue that can promote N-acetylation.(A) D HC -aF comprises ppaF with the hydrophobic core of the signal sequence replaced with that of DPAP B, creating an SRP-dependent substrate.D HC -aF with the endogenous P2 residue (MR) or with a serine inserted at position 2 (MS) were translated in vitro in a yeast extract supplemented with [ 35 S] methionine in the presence or absence of yeast microsomes (yRM).Translated proteins were immunoprecipitated with anti-aF antibodies prior to analysis by SDS-PAGE and phosphorimaging.Positions of the unprocessed (D HC aF) and glycosylated (g-D HC aF) forms of the protein are indicated.(B) WT and MS ppaF were translated in yeast extract in the presence of [ 35 S] methionine and incubated with or without yeast microsomes.(TIF) Figure S5 Peak hydrophobicity analysis of Yeast Signal Sequences.Mean peak hydrophobicity of yeast signal sequences group according to their predicted N-terminal processing.Peak hydrophobicity determined based on Kyte-Doolittle [57] with awindow size of 11.The ''acetylated,'' ''methionine cleaved not acetylated,'' and ''non-processed'' groups had mean peak hydrophobicities of 2.59360.0657(SEM), 2.51860.0673,and 2.33360.0352,respectively.The ''acetylated'' and ''cleaved not acetylated'' groups differed significantly from the ''unprocessed'' group (p,0.01 and p,0.05, respectively, one-way ANOVA with Tukey's multiple comparison test).The acetylated and cleaved group were not significantly different.Note that only two signal sequences of the acetylated group (,6%) had a peak hydrophobicity of less than 2, the threshold for interaction with SRP [30].(TIF)