title: Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane.
abstract: Nα-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling,in vitroassays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C terminus was unstructured in solution and only folded into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 toward membranes containing the phosphatidylinositol PI(4)P, thus possibly explaining the primary residency of Naa60 at the PI(4)P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.
authors.forename: Henriette
authors.initials: H
authors.lastname: Aksnes
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.
authors.forename: Marianne
authors.initials: M
authors.lastname: Goris
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.
authors.forename: Øyvind
authors.initials: Ø
authors.lastname: Strømland
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.
authors.forename: Adrian
authors.initials: A
authors.lastname: Drazic
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.|the Computational Biology Unit, Department of Informatics, University of Bergen, N-5020 Bergen, and.
authors.forename: Qaiser
authors.initials: Q
authors.lastname: Waheed
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen.|the Computational Biology Unit, Department of Informatics, University of Bergen, N-5020 Bergen, and.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Reuter
authors.affiliation: From the Department of Molecular Biology, University of Bergen, N-5020 Bergen, thomas.arnesen@uib.no.|the Department of Surgery, Haukeland University Hospital, N-5021 Bergen, Norway.
authors.forename: Thomas
authors.initials: T
authors.lastname: Arnesen
mesh_terms: D002151:Calorimetry; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000201:enzymology; D004252:DNA Mutational Analysis; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D063214:N-Terminal Acetyltransferase F / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D012270:Ribosomes / Q000737:chemistry; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; C570958:NAA60 protein, human; D010455:Peptides; D049452:Green Fluorescent Proteins; D014364:Tryptophan; D063214:N-Terminal Acetyltransferase F
keywords: Golgi; N-terminal acetylation; NAT; Naa60; NatF; PI(4)P; acetylation; acetyltransferase; lipid binding protein; membrane enzyme