Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Abstract

We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10Δ cells, which lack the NatA N α -terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10Δ cells by the Arg/N-end rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-end rule pathway in naa10Δ cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-end rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10Δ cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-end rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-end rule pathway.roteins called chaperones promote the in vivo protein folding and counteract misfolding, maladaptive protein aggregation, and other perturbations of proteostasis (1-4). Hsp90 is an ATPdependent chaperone system that mediates the conformational maturation and stabilization of many proteins. Referred to as Hsp90 clients, these proteins include kinases, transcriptional regulators, and many other proteins that comprise at least 20% of a cellular proteome. The diversity of Hsp90 clients and the broad range of processes that involve Hsp90 underlie its necessity for cell viability in all eukaryotes (5-11). Cochaperones of the ∼90-kDa Hsp90 comprise, in mammals, ∼30 distinct proteins. They participate in the delivery of clients to Hsp90 and contribute to related processes, including ATP hydrolysis by Hsp90 (12). Because of its ability to modulate protein conformations, the Hsp90 system can either exacerbate or counteract the fitness-reducing effects of mutations in cellular proteins. This property of Hsp90 affects the standing genetic variation and thereby influences the impact of natural selection on rates and directions of evolution (13,14).The N-end rule pathways comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, thereby causing the degradation of these proteins by the proteasome or autophagy in eukaryotes and by the proteasome-like ClpAP protease in bacteria (Fig. 1) (15-26). The main determinant of an N-degron is a destabilizing Nt-residue of a protein. Many N-degrons become active through their enzymatic Nt-acetylation, Ntformylation...

Emerging branches of the N-end rule pathways are revealing the sequence complexities of N-termini dependent protein degradation

“…This apparent dichotomy may be rationalized in several ways, which include sufficient residual acetylation in the mutant, additional unforeseen sequence specificity of the Ac-N-end rule pathway or that many of the N-termini are inaccessible for the Ac-N-end rule pathway as this pathway has been reported to degrade protein subunits that are in excess of their target complex (Shemorry et al 2013). In support of a model where many of these acetylated N-termini may not be directly accessible for the Ac-N-end rule pathway is the recent report that demonstrates this pathway is involved in the degradation of several HSP90 client proteins (Oh et al 2017). While much is left to be explored regarding this interplay between N-terminal acetylation and protein stability, some of its implications and details have been discussed in detail (Eldeeb and Fahlman 2016a;Lee et al 2016).…”

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+biosynthetic enzymes inSaccharomyces cerevisiae

“…Nascent Nma1 and Nma2 are substrates of Hsp70 and other chaperones [60,61], and it is possible that Nt-acetylation promotes this interaction. In line with this, Nt-acetylation of Hsp90 subunits is required to mediate interactions with its substrates, and absence of Nt-acetylation increases degradation of both Hsp90 subunits and its substrates [33]. Weakened Nma1 and Nma2 interaction with chaperones is likely to increase misfolding of these proteins possibly leading to cotranslational degradation.…”

“…This step was skipped if measuring basal protein level. Cell lysates for Western blot were made as described [33]. Briefly, 1 A 600 unit of c ells was pelleted and supernatant was removed.…”

“…In absence of Nt-acetylation, Ac/N-end rule proteins may become unrecognizable by their E3 ligases and long-lived [31]. While Nma1 and Nma2 do not fulfill the requirements for degradation by the Arg/N-end rule pathway, it is worth noting that examples of Ac/Nsubstrates becoming Arg/N-substrates in the absence of Nt-acetylation have been observed [32,33]. In addition to the possibility of being degraded by the N-end rule pathways, non-Nt-acetylated Nmnats may be degraded during translation since the absence of Nt-acetylation may affect its folding or complex formation.…”

Nα-terminal acetylation of proteins by NatA and NatB serves distinct physiological roles inSaccharomyces cerevisiae

“…Third, Nt-acetylation has been connected to protein folding and stability since it reduces the Nterminal charge and thereby can stabilize N-terminal α-helices of proteins such as mitochondrial chaperonin 10 (Cpn10; Jarvis et al, 1995;Ryan et al, 1995), tropomyosin (Greenfield et al, 1994) and α-synuclein (Dikiy & Eliezer, 2014;Bartels et al, 2014). Finally, a number of publications link Ntacetylation to protein degradation in yeast (Oh et al, 2017;Dörfel et al, 2017;Holmes et al, 2014;Zattas et al, 2013;Pezza et al, 2009). There, acetylated N-termini form an N-degron that targets proteins via the Ac/N-end rule pathway for ubiquitination by E3 ubiquitin ligases including Not4 and Doa10, followed by proteasomal degradation (Hwang et al, 2010;Shemorry et al, 2013).…”