Specificity of cotranslational amino-terminal processing of proteins in yeast

Abstract

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.

Multiplicity of the β Form of the cAMP-dependent Protein Kinase Inhibitor Protein Generated by Post-translational Modification and Alternate Translational Initiation

“…. ), which is unfavorable for such amino-terminal processing (15,16). The location of PKI␤-78 on the two-dimensional gel, in comparison with that of PKI␤-70, is consistent with the difference between them.…”

“…For the anti-PKI␣-(5-22) and anti-PKI␤- (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) antibodies, the conjugates were synthesized by derivatization of the amino terminus of the synthesized peptide with the heterobifunctional cross-linker maleimidohexanoyl-N-hydroxysuccinimide ester and then cross-linking to N-succinimidyl S-acetylthioacetate-derivatized ovalbumin (20,21). For the anti-PKI␤-(60 -70) antibodies, the peptide was synthesized with an additional carboxylterminal cysteine, and the conjugates were synthesized by cross-linking maleimidohexanoyl-N-hydroxysuccinimide ester-derivatized ovalbumin to the peptide via the terminal cysteine.…”

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

“…Third, the steady-state size of SDS-resistant Sup35 aggregates increases in [PSI ϩ ] strains deficient in Hsp104, Hsp70, or Hsp70 cochaperones (Eaglestone et al, 2000;Wegrzyn et al, 2001;Cox et al, 2003;Kryndushkin et al, 2003;Jones et al, 2004;Song et al, 2005;Fan et al, 2007;Kryndushkin and Wickner, 2007;SatputeKrishnan et al, 2007;Sadlish et al, 2008), an observation that is completely opposed to our findings for NatA mutants ( Figure 5C). In addition to these factors, the ubiquitin-conjugating enzyme Ubc4 and the Hsp70 family members Ssb1 and Ssb2 are predicted or proven substrates for NatA (Huang et al, 1987;Polevoda et al, 1999), are modifiers of the [PSI ϩ ] prion cycle Allen et al, 2007), and have demonstrated synthetic interactions with NatA (Gautschi et al, 2003;Pan et al, 2006). Despite these intriguing connections, ⌬ubc4 and ⌬ssb1⌬ssb2 strains, unlike NatA null strains, display the [PSI ϩ ] nonsense suppression phenotype, indicating that they are unlikely to be the crucial NatA targets.…”

“…To further explore a potential role for Hsp70 N-terminal acetylation in modulating the [PSI ϩ ] phenotype, we next generated a ssa1 allele containing a Ser-to-Pro substitution at the second position, a change that has been shown to block N ␣ -acetylation by NatA in other proteins (Huang et al, 1987;Geissenhoner et al, 2004). To rule out the possibility that acetylated Ssa2, which is also expressed under normal growth conditions (Werner-Washburne et al, 1989), could mask any potential effects of unacetylated Ssa1(S2P) on the [PSI ϩ ] phenotype, we also disrupted SSA2 in both wild-type and ssa1S2P strains.…”

“…By immunoblotting, Hsp70 constitutively expressed from the SSA1 and SSA2 loci accumulated to similar levels in wild-type and NatA mutants, whereas expression of the stress-inducible forms of Hsp70 from the SSA3 and SSA4 loci were not detected in either wild-type or NatA strains under normal growth conditions (Supplemental Figure S4), indicating that loss of NatA activity did not alter Hsp70 expression profiles. To further explore a potential role for Hsp70 N-terminal acetylation in modulating the [PSI ϩ ] phenotype, we next generated a ssa1 allele containing a Ser-to-Pro substitution at the second position, a change that has been shown to block N ␣ -acetylation by NatA in other proteins (Huang et al, 1987;Geissenhoner et al, 2004). To rule out the possibility that acetylated Ssa2, which is also expressed under normal growth conditions (Werner-Washburne et al, 1989), could mask any potential effects of unacetylated Ssa1(S2P) on the [PSI ϩ ] phenotype, we also disrupted SSA2 in both wild-type and ssa1S2P strains.…”