N-terminal-acetylases (NATs) catalyse the covalent attachment of an acetyl moiety from acetyl-CoA to the free alpha-amino group at the N-terminus of a protein. This irreversible modification neutralizes the positive charge at the N-terminus, makes the N-terminal residue larger and more hydrophobic, and prevents its removal by hydrolysis. NatF is found only in higher eukaryotes, and is absent from yeast. Unlike other Nat systems the enzyme is located in the Golgi apparatus. It faces the cytosolic side of intracellular membranes, and specifically acetylates transmembrane proteins whose N termini face the cytosol. NatF targets N-terminal L-methionine residues attached to Lys, Ser, Val, Leu, Gln, Ile, Tyr and Thr residues.
N-terminal-acetylases (NATs) catalyse the covalent attachment of an acetyl moiety from acetyl-CoA to the free alpha-amino group at the N-terminus of a protein. This irreversible modification neutralizes the positive charge at the N-terminus, makes the N-terminal residue larger and more hydrophobic, and prevents its removal by hydrolysis. NatF is found only in higher eukaryotes, and is absent from yeast. Unlike other Nat systems the enzyme is located in the Golgi apparatus. It faces the cytosolic side of intracellular membranes, and specifically acetylates transmembrane proteins whose N termini face the cytosol. NatF targets N-terminal L-methionine residues attached to Lys, Ser, Val, Leu, Gln, Ile, Tyr and Thr residues.
Naa60 attaches to membranes of the Golgi apparatus. Molecular determinants for the interaction of Naa60 with the membrane by means of computational modeling, in vitro liposome assays including intrinsic tryptophan fluorescence, circular dichroism, and isothermal titration calorimetry (ITC), and mutational analysis combined with localization determination in cellulo. Model of Naa60 membrane association and N-terminal acetylation at the membrane, overview. Peripheral binding to Golgi-like liposomes. Naa60 has a preference for phosphatidylinositol 4-phosphate which may recruit it to the Golgi membrane mainly through Arg212 and Arg215 of Pred-alpha2, and through Lys233 and Arg240 of the C-terminal tail
the C-terminal tail of Naa60 (amino acids 182-242), which constitutes an extra segment, not present in the other catalytic Naas, is defined as the part providing organellar localization. Human eGFP-tagged Naa60 displays an organellar localization recombinantly expressed in Saccharomyces cerevisiae, and this localization pattern is lost and replaced by a cytosolic signal upon truncation of the last 61 amino acids. Naa60 uses a membrane targeting mechanism that is also functional in fungi, although Naa60 is not found in this kingdom. After incubation of the Naa60(189-242) peptide with Golgi-like liposomes composed of 52% phosphatidylcholine, 20% phosphatidylethanolamine, 15% cholesterol, 8% phosphatidylinositol 4-phosphate, and 5% phosphatidylserine, a clear blue shift from 355 to 348 nm is observed, hence indicating an interaction between Naa60(189-242) and these liposomes, residue Trp227 dips into the membrane
in complex with acetyl-coenzyme A or coenzyme A, hanging drop vapor diffusion method, using 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% (v/v) glycerol, 3% (v/v) Tacsimate pH 4.0, and 7.5% (w/v) polyethylene glycol 3350
the mutant shows an approximate 2.5 to 4fold reduction in kcat and a small increase in KM value, so the catalytic efficiency of this mutant is approximately 3fold of the wild type enzyme
site-directed mutagenesis, enzyme deletions suggest membrane interaction by two regions matching the predicted helices (Pred), hydrophobic amino acids in Pred-alpha are mutated. The I190A/L191A/Y193A/I194A mutation, which involves two of the buried amino acids from Pred-alpha1, causes a mixed organellar and cytosolic localization in contrast to the wild-type. This mutation either causes only partially impaired membrane integration of Pred-alpha1 or additional segments are able to maintain some membrane association without Pred-alpha1. Truncation and deletion constructs of eGFP-tagged Naa60 demonstrate regions in the C-terminal tail important for membrane association in cellulo
site-directed mutagenesis, the mutation results in a mixed localization of the enzyme, with a partial and almost completely extractable organellar fraction, but with a more severe degree of mislocalization compared with mutant I190A/L191A/Y193A/I194A
generation of truncated enzyme fragment Naa60(189-242), which peripherally binds Golgi-like liposomes possibly with a horizontal orientation of helices relative to the membrane bilayer
the mutant has a 4-5fold increase in kcat but at the same time a 5-10fold increase in KM value , so the catalytic efficiency of this mutant is approximately 0.5fold of the wild type enzyme
development of a proteolysis-based assay, named PROMPT, i.e. PROtease assay for Membrane Protein Topology, to determine the topology of protein C-termini