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

•Naa60 is an organelle N-terminal acetyltransferase, and it acts on the cytosolic face•Most transmembrane proteins are Nt-acetylated, and Naa60 acts specifically on these•Naa60 mainly localizes to the Golgi and is essential for Golgi ribbon structure•PROMPT, a novel assay for membrane topology of proteins, is presented

SummaryN-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular 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 transmembrane 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 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.

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