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

Abstract

N-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.

The biological functions of Naa10 — From amino-terminal acetylation to human disease

“…In line with this, systematic bioinformatic studies of predicted N-terminal processing found that cytoplasmic proteins are typically biased in favor of acetylation, whereas secretory proteins are not (Forte et al, 2011). A differential N-terminal combined fractional diagonal chromatography (COFRADIC) study on cytosolic and organellar fractions of A-431 cells revealed that the degree of NTA is lower for integral membrane proteins compared to their soluble counterparts, supporting this idea (Aksnes et al, 2015c). Furthermore, mutation of the second amino acid of the post-transcriptionally targeted proteins CPY (carboxypeptidase Y), Pdi1 and ppα-factor that allowed acetylation by NatA inhibited their translocation, whereas this effect was restored in NAA10 Δ cells (Forte et al, 2011).…”

“…Furthermore, no disruption of the nuclear membrane, endoplasmic reticulum, Golgi apparatus, mitochondria, or bud neck was observed upon NatC deletion, suggesting the intactness of these organelles and subcellular structures as judged by the unchanged shape, number, size and distribution in the cell (Aksnes et al, 2013). A follow-up study showed that not NatC but rather NatF (Naa60) is associated with Golgi membranes and the ER, and disruption of this Naa60 induces Golgi fragmentation (Aksnes et al, 2015b). Taken together, this indicates that NatC is not – at least not in general – a determinant for substrate subcellular localization (Aksnes et al, 2013).…”

“…For instance it has been shown that the majority of hNatA is non-polysomal (Arnesen et al, 2005a) and a minor fraction of cytosolic hNaa10 exists independent of the NatA complex, carrying out post-translational acetylation as mentioned above (Van Damme et al, 2011b). Mammalian Naa10, Naa11 and Naa15 and Naa50 have been reported to be mainly localized in the cytoplasm and to a lesser extent to the nucleus (Fluge et al, 2002; Sugiura et al, 2003; Bilton et al, 2005; Arnesen et al, 2006a,b; Chun et al, 2007; Xu et al, 2012; Park et al, 2014; Zeng et al, 2014; Aksnes et al, 2015c). In mouse, an isoform specific localization of Naa10 has been described.…”

Actin polymerization and cell motility are affected by NAA80-mediated posttranslational N-terminal acetylation of actin

“…NAA80/NatH differs in that Nt-acetylation of actin occurs posttranslationally since it constitutes the final step of a unique step-by-step mechanism for Nt-maturation. Posttranslational activity is also suggested for the other more recently identified NATs, NAA60/NatF and NAA70/NatG due to their subcellular localization [8,9]. Contrary to the typical NAT, NAA80/NatH appears to be highly selective with actin as the only substrate in vivo in animals, as demonstrated by the lack of actin Nt-acetylation in NAA80 KO cells by COFRADIC analysis [6].…”

(Hyper)tension release by N-terminal acetylation

“…Nt-acetylation was recently described to be common also among transmembrane proteins [10]. This part of the Ntacetylome and the functions of its Nt-acetylation are largely unexplored and future studies might reveal Ac/N-degrons in transmembrane proteins.…”