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Ribosome-associated chaperones are found in all organisms but exhibit different structures and mechanisms. In eukaryotes such as the yeast Saccharomyces cerevisiae, two ribosome-associated chaperones, RAC (ribosome-associated complex) and NAC (nascent polypeptide-associated complex), interact (cotranslationally) with nascent proteins (5), while downstream folding support is provided by different cytosolic chaperones for proteins that require additional assistance (figure 1 adopted from 6). Moreover, about one third of newly synthesized proteins are cotranslationally transported to other cellular destinations such as the endoplasmic reticulum, indicating that, in addition to chaperones, targeting factors also interact at an early stage with nascent polypeptides (4). Indeed, it has been shown that the SRP (signal recognition particle), which facilitates delivery of secreted proteins at the endoplasmic membrane, associates with hydrophobic targeting signals present in secretory proteins as they emerge from the ribosome (7). Furthermore, many newly synthesized proteins are cotranslationally modified such as removal of the N-terminal methionine or acylation of the N-terminus. This raises the question how these different factors are accommodated by the ribosome. Biochemical and structural data have established that the ribosome is equipped with a multiple factor-docking platform located around the tunnel exit (3). It comprises ribosomal proteins (eL19, eL22, uL22, uL23, uL29, eL31 and eL39) and rRNA elements and serves as binding site for chaperones, processing enzymes and targeting factors. Specifically, uL23 functions as multifunctional docking site and coordinates the binding of NAC and SRP (3). Recently, detailed structures of a eukaryotic ribosome containing either NAC or NAC and SRP were presented (8), providing important mechanistic insight into how NAC modulates SRP function and thereby controls specificity during protein targeting. Here, I will discuss these structures as well as their current understanding.

The ribosome and ribosome-associated chaperones

The ribosome is a large ribonucleoprotein particle that is responsible for decoding mRNA into protein. This is known as translation and represents the final step of the central dogma. In bacteria, the ribosome comprises three rRNA molecules and about 55 proteins with a mass of approximately 2.4 MDa (3,9). The yeast ribosome is ~4 MDa and is made up of four rRNA molecules and about 80 proteins (3,10). Structurally, the ribosome consists of two subunits – a large and small subunit of 30S and 50S in bacteria and 40S and 60S in eukaryotes, respectively (figure 2 adapted from 3). The ribosome is equipped with four functional units, enabling the decoding of mRNA into protein (3). The first of these is the so-called decoding center that is responsible for selection of the proper aminoacyl-tRNA as specified by the mRNA’s codon. The decoding center (DC) is located in the small subunit (figure 2) and recognizes the architecture codon-anticodon base pairing. The peptidyl-transferase center (PTC) is the second feature and represent the ribosome’s catalytic core. It is the site of peptide bond formation and resides in the large subunit. The PTC is entirely made up of rRNA, indicating that the peptidyl transferase activity is not facilitated by ribosomal proteins but only by rRNA (11). The ribosomal tunnel inside the large subunit is the third functional feature and connects the PTC with the ribosomal exit site.

The tunnel has a length of 80-100 Å and a width ranging from 10 Å at its narrowest point to 20 Å at its widest point. The tunnel wall is primarily made up of rRNA, although two proteins (uL4 and uL22) are part of the tunnel wall as well (3). The tunnel does not represent a passive channel for nascent chains but plays an active role in protein folding. It was recently shown that small domains can fold while still inside the tunnel. In fact, it was found that the tunnel accelerates folding and stabilizes the folded state (12). The fourth functional unit comprises the multiple factor-docking platform. This is located around the tunnel exit and is made up of ribosomal proteins (eL19, eL22, uL22, uL23, uL29, eL31 and eL39) and rRNA. It has been established that it serves as binding site for chaperones, processing enzymes and targeting factors. For example, uL23 associates with NAC, SRP and Sec61 and it is therefore known as the universal ribosome docking site (3,5). Additionally, eL19, eL22, uL22, uL29, eL31, eL39 as well as rRNA elements also contribute to the binding of ribosome-associated factors. These different factors play a crucial role in the early life of newly synthesized proteins. Targeting factors such as SRP ensure cotranslational delivery of secretory proteins at the endoplasmic membrane, while processing enzymes cotranslationally modify the N-terminus of proteins. Ribosome-associated chaperones are found in all organisms and control the early folding steps during translation. Remarkably, these chaperones exhibit distinct structures and utilize differing mechanisms. The biological and structural features of NAC are described below in more detail.

Biological features of NAC

NAC was first described in 1994 as a factor that initially interacts with nascent polypeptides as they emerge from the ribosome, thereby preventing binding of SRP to proteins devoid of a signal peptide (13). NAC is a dimeric protein that is present in all eukaryotes as well as archaea (figure 3 adopted from 5). In contrast to eukaryotes, in which NAC forms a heterodimer made up of an α (α NAC) and β subunit (βNAC), archaeal NAC is a homodimer formed by two α subunits. Structurally, both subunits contain a so-called NAC domain that folds into a six-stranded β-barrel-like structure and enables dimerization of both subunits (5,6). Furthermore, α NAC contains a C-terminal ubiquitin-associated domain (UBA) that adopts a compact three-helix bundle. This domain is not present in βNAC and its function is not well understood. NAC is expressed at relatively high levels and associates with ribosomes in a 1:1 stoichiometry. βNAC is probably responsible for association with the ribosome because it contains a conserved ribosome-binding motif at its C-terminus and deletion of this domain or replacement of its core residues prevents ribosome binding. Evidence obtained by crosslinking experiments indicates that βNAC associates with the ribosomal protein uL23 and possibly also eL31, while α NAC may interact with uL29 and uL23, respectively (5,6). The function of NAC is as yet not well understood but deletion of NAC results in embryonic death in mice, worms and fruit flies, thereby pointing towards a crucial cellular role in higher eukaryotes (5,6).

Different functions of NAC have been proposed. For example, a role as cotranslational chaperone was suggested for NAC based on its association with the ribosome as well as its interaction with nascent polypeptides (13,14). Analysis of NAC’s cotranslational specificity showed that it interacts with nearly all newly synthesized proteins but, strikingly, αNAC and βNAC recognize different subsets of nascent polypeptides probably through their flexible N-terminal regions (15). In addition to its role as early acting chaperone, NAC is also thought to modulate protein targeting. For example, it was reported that NAC has a primary function in regulating the cotranslational transport to the ER and it initiates the cotranslational targeting of mitochondrial precursor proteins through promoting the interaction of ribosomes with the mitochondrial surface (16). Interestingly, biochemical evidence showing that the N-terminal tail of βNAC inserts deep into the ribosomal tunnel to scan polypeptide substrates during biogenesis close to the PTC was recently provided (17). This monitoring of nascent polypeptides inside the ribosomal tunnel is critical for proper protein trafficking within the cell. Moreover, it was found that association of NAC with ribosomes was needed to prevent incorrect ribosome-Sec61 interactions, thereby indicating that NAC negatively regulates the binding of ribosomes to Sec61 to maintain ER targeting specificity (18,19). NAC probably also serves as negative regulator of SRP because it allows ribosome-binding of the SRP only if a signal sequence emerges from the ribosomal tunnel (13). The activity of NAC, therefore, opposes that of SRP and the simultaneous binding of these factors to the ribosome suggests that signal sequences are handed over from NAC to SRP. Furthermore, a ribosome-independent chaperone function for NAC was reported recently (20). This was based on the finding that in higher eukaryotes a significant fraction of NAC is not bound to ribosomes, while the ribosome-binding domain is essential for the chaperone activity of NAC. Another ribosome-independent function of NAC concerns ribosome biogenesis within the nucleus (21), while a role of NAC in protein aggregation, disaggregation as well as the removal of aggregates was also reported (20). The evidence thus far suggests that NAC is a versatile protein biogenesis factor that is able to function both in conjunction with the ribosome or separately. Within the context of the ribosome, NAC functions as a cotranslational chaperone that controls the early folding of nascent polypeptides and acts as negative SRP regulator, thereby fine-tuning ER targeting specificity. In the absence of ribosomes, NAC probably represents a general holdase that maintains substrates in a soluble state thereby allowing their refolding, and is active within the nucleus as ribosome biogenesis factor.

Structural features of NAC

NAC was initially identified as a factor that associates with nascent polypeptides that emerge from the ribosome unless an ER signal sequence is fully exposed (13). This implies that NAC prevents SRP-mediated mistargeting of nascent polypeptides that lack a signal sequence. Both SRP and NAC bind to uL23 near the ribosomal exit site and are therefore ideally positioned to interact with nascent polypeptides that emerge from the ribosome (4,5). Conceivably, both NAC and SRP bind simultaneously to ribosomes. The interplay between NAC and SRP was explored in more detail employing biochemical in vitro experiments (22), showing that NAC binds to nascent polypeptides of secretory proteins but is displaced by SRP upon emergence of a signal sequence from the ribosomal tunnel. This points towards a handoff intermediate in this NAC to SRP exchange. Translating ribosomes are thus able to recruit both NAC and SRP. Upon further elongation, however, NAC reassociates with SRP-containing translating ribosomes. These findings reveal a functional interplay between NAC and SRP whereby both factors bind simultaneously to different stretches of a nascent polypeptide and NAC serves as negative SRP regulator. How this interplay is structurally coordinated is, however, unclear. Recently, detailed structures of a translating ribosome containing these factors were presented (8). To this end, complexes were reconstituted in vitro through mixing ribosomes translating a secretory protein with NAC and SRP followed by cryo-EM analysis. Two complexes were observed, namely: (i) a complex probably corresponding to the state before cargo transfer comprising a translating ribosome bound to NAC and (ii) a complex most likely representing the state after cargo transfer made up of a translating ribosome containing both NAC and SRP. Conceivably, NAC initially binds to translating ribosomes that display a signal sequence or not, while transfer to SRP only occurs after the signal sequence has fully emerged from the ribosomal exit. Figure 4 shows the structure of the NAC-containing ribosome at 2.9 Å in surface or ribbon representation. The middle panel (all ribosomal proteins are omitted for clarity) shows that both subunits are well resolved (with αNAC in purple and βNAC in yellow). The globular domain of both subunits comprises ß-strands and anti-parallel helices. This domain facilitates subunit dimerization. Moreover, the structure shows the interaction between the ribosome and the N-terminal tail of βNAC. The structure of a single NAC protomer is presented in the right panel (with αNAC in purple and βNAC in yellow). βNAC’s N-terminal tail contains a positively charged motif followed by a helix and a loop. This loop is folded into an anchor-shaped turn that wraps around eL22 and contacts eL19 and rRNA. The globular domain also contributes to ribosome binding. In fact, two positively charged helices of both subunits associate with rRNA. Furthermore, the binding site of NAC’s globular domain on the ribosome overlaps with that of SRP. This suggests that the binding of NAC at the ribosomal exit site is the basis of SRP inhibition, which is most likely relieved when a signal sequence emerges from the ribosome. Conceivably, emergence of a signal sequence weakens the interaction between the ribosome and the globular domain of NAC. This implies that NAC’s globular domain controls SRP access. Indeed, biochemical binding studies confirmed that emergence of a signal sequence from the ribosomal exit weakens the interaction of the globular domain with the ribosome, while crosslinking experiments confirmed that a signal sequence destabilizes the NAC globular domain. The N-terminal tail of βNAC remains, however, bound to the ribosome. These data, therefore, point towards a remodeling of the interactions between NAC and the ribosome following emergence of a signal sequence.

The structure of the ribosome bound to SRP and NAC is presented in figure 5 at 2.8 Å resolution in surface representation. SRP (shown in green), comprising SRP19, SRP68 and SRP54, is well resolved. In contrast, the globular domain of NAC (with αNAC in pink and ßNAC in yellow) is not observed because its place at the ribosome is occupied by the M-domain of SRP54 (right panel with all ribosomal proteins omitted for clarity and the nascent secretory polypeptide in red). Furthermore, the flexible UBA domain of αNAC (in pink) is associated with the M-domain of SRP54 through salt bridges and hydrogen bonds between conserved residues. The UBA-binding site on SRP54, moreover, overlapped with that of the SRP receptor (SR), thereby raising the possibility that formation of the SRP-SR complex displaces NAC from SRP at the endoplasmic membrane. The interaction of NAC’s UBA domain and SRP’s M-domain stabilizes their simultaneous binding on a translating ribosome. This interaction occurs before docking of the SRP and subsequent transfer of the signal sequence. The association between NAC’s UBA domain and SRP’s M-domain is crucial for the proper targeting of secretory proteins to the endoplasmic membrane. NAC is bound to most ribosomes at the start of translation through a high affinity anchor and a weakly bound globular domain that blocks SRP access to nascent polypeptides.