Investigating the functionality of a ribosome-binding mutant of NAA15 using Saccharomyces cerevisiae

Abstract

ObjectiveN-terminal acetylation is a common protein modification that occurs preferentially co-translationally as the substrate N-terminus is emerging from the ribosome. The major N-terminal acetyltransferase complex A (NatA) is estimated to N-terminally acetylate more than 40% of the human proteome. To form a functional NatA complex the catalytic subunit NAA10 must bind the auxiliary subunit NAA15, which properly folds NAA10 for correct substrate acetylation as well as anchors the entire complex to the ribosome. Mutations in these two genes are associated with various neurodevelopmental disorders in humans. The aim of this study was to investigate the in vivo functionality of a Schizosaccharomyces pombe NAA15 mutant that is known to prevent NatA from associating with ribosomes, but retains NatA-specific activity in vitro.ResultsHere, we show that Schizosaccharomyces pombe NatA can functionally replace Saccharomyces cerevisiae NatA. We further demonstrate that the NatA ribosome-binding mutant Naa15 ΔN K6E is unable to rescue the temperature-sensitive growth phenotype of budding yeast lacking NatA. This finding indicates the in vivo importance of the co-translational nature of NatA-mediated N-terminal acetylation.

Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency

“…NAA15 anchors the NatA complex to the ribosomal large subunit via RPL25 (L23A). 55 – 58 , 64 Consistent with this model, some of these affected proteins clustered near the NAA15/NatA docking site. Presumably, NAA15 binding to one or more of these proteins 56 protects them from subsequent degradation either during ribosome assembly or subsequent ribosomal activity.…”

“…NAA15 facilitates binding of the NatA complex to the ribosome. 55 – 58 The general docking site for NatA resides at the ribosomal exit tunnel near large ribosomal unit RPL25 (L23A), a general docking platform for various factors that are transiently associated with ribosomes. 55 , 57 , 59 – 61 Dysregulated ribosomal proteins observed in both NAA15 +/− and NAA15 −/− iPSCs clustered near the NatA docking site (Figure 5 A through 5 C).…”

“…By contrast, levels of 562 proteins were altered in both NAA15-null and NAA15-haploinsufficient cells suggesting a possible role for NAA15 and NatA in translation efficiency and protein stability in addition to Nt-acetylation. If NAA15 was the only available ribosome anchor for NAA10 and the NatA complex in human cells as it is in yeast cells, 8 , 55 , 58 NAA15 null cells should present with a complete lack of Nt-acetylation of all NatA substrates. The explanation for the partial effect observed may be due to the presence of the NAA15 paralogue NAA16 in human cells; however, the low expression of NAA16 in iPSCs suggests the need for additional studies to measure whether the paralogue to NAA15 had any effect on N-terminal acetylation.…”

Co-translational, Post-translational, and Non-catalytic Roles of N-Terminal Acetyltransferases

“…NatA was first discovered in Saccharomyces cerevisiae and is composed of the catalytic subunit NAA10 and the auxiliary subunit NAA15 (Figure 2, column 2; Arnesen et al, 2005a;Mullen et al, 1989;Park and Szostak, 1992). NAA15 is essential for NatA activity in two ways: it acts as a ribosomal anchor via contacts to the ribosome (Gautschi et al, 2003;Magin et al, 2017;Varland and Arnesen, 2018), and it modulates the substrate specificity of NAA10 (Liszczak et al, 2013). Although monomeric NAA10 may target acidic amino acids (at least in vitro), the bulk of cellular NAA10 is bound to NAA15, and this complex prefers substrate N termini starting with small amino acids like Ser and Thr (Liszczak et al, 2013;Van Damme et al, 2011a).…”

Naa12compensates forNaa10in mice in the amino-terminal acetylation pathway

“…Across species, Naa10 is bound to its auxiliary subunit, Naa15, which links the catalytic subunit to the ribosome to facilitate co-translational Nt-acetylation of proteins as they emerge from the exit tunnel (23,(49)(50)(51)(52)(53). Due to its high sequence similarity (see also S4B Fig), we suspected that Naa12 may also interact with Naa15.…”