An Organellar Na-Acetyltransferase, Naa60, Acetylates Cytosolic N Termini of Transmembrane Proteins and Maintains Golgi Integrity.pptx

Graphical Abstract Highlights d Naa60 is an organelle N-terminal acetyltransferase, and it acts on the cytosolic face d Most transmembrane proteins are Nt-acetylated, and Naa60 acts specifically on these d Naa60 mainly localizes to the Golgi and is essential for Golgi ribbon structure d PROMPT, a novel assay for membrane topology of proteins, is presented In Brief Aksnes et al. show that N-terminal acetylation, a common modification of soluble eukaryotic proteins, is also frequent among transmembrane proteins. They find Naa60 to be an organelle-associated N-terminal acetyltransferase, with cytosolic activity toward N termini of transmembrane proteins, likely involved in the maintenance of the Golgi's structural integrity. SUMMARY N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltrans-ferases (NATs). NatF, or Na-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicel-lular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis 2011). Although the overall biological function of this ubiquitous and cotranslational process has been difficult to pinpoint, several reports suggest its impact on protein subcellular locali-zation (Behnia et al. NatF (Naa60) is the most recently identified member of the NAT family and was shown to be conserved among multicellular eukaryotes while absent from unicellular eukaryotes such as yeast (Van Damme et al., 2011b). NatF contributes to an evolutionary shift toward increased N-terminal acetylation (Nt-acety-SUMMARY N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltrans-ferases (NATs). NatF, or Na-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicel-lular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates trans-membrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi's structural integrity. This work identifies a NAT associated with mem-branous compartments and establishes N-terminal acetylation as a common modification among trans-membrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.