An organellar Nα-acetyltransferase Naa60 acetylates cytosolic N-termini of transmembrane proteins and maintains Golgi integrity

N-terminal acetylation is a major and vital protein modification catalysed by N-terminal acetyltransferases (NATs). NatF or Nα-acetyltransferase 60 (Naa60) was recently identified as a NAT in higher eukaryotes. Here, we found 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 Naa60 to face the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60 knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins, and that is has a preference for N-termini facing the cytosol. Moreover, NAA60 knockdown caused Golgi fragmentation, indicating an important role in maintenance of Golgi structural integrity. This work uncovers the first NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus far poorly characterized part of the N-terminal acetylome.