Chaperone Interactions at the Ribosome

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Elke Deuerling, Martin Gamerdinger, and Stefan G. Kreft

Molecular Microbiology, Department of Biology, University of Konstanz, 78464 Konstanz, Germany Correspondence: [email protected]

The continuous refreshment of the proteome is critical to maintain protein homeostasis and to adapt cells to changing conditions. Thus, de novo protein biogenesis by ribosomes is vitally important to every cellular system. This process is delicate and error-prone and requires, besides cytosolic chaperones, the guidance by a specialized set of molecular chaperones that bind transiently to the translation machinery and the nascent protein to support early folding events and to regulate cotranslational protein transport. These chaperones include the bacterial trigger factor (TF), the archaeal and eukaryotic nascent polypeptide-associated complex (NAC), and the eukaryotic ribosome-associated complex (RAC). This review focuses on the structures, functions, and substrates of these ribosome-associated chaperones and highlights the most recent findings about their potential mechanisms of action.

he life of every protein, irrespective of its or/and cleavage of the amino-terminal methio- Tfunction or origin, starts by its messenger nine. Thus, different factors act cotranslationally RNA (mRNA)-templated translation on ribo- on nascent chains assumingly in a highly specific somes. Upon synthesis by ribosomes, the emerg- and coordinated manner, both temporally and ing polypeptide chains directly start their fold- spatially, to ensure the functionality of the trans- ing program into the unique three-dimensional latome (Kramer et al. 2009; Gamerdinger 2016). (3D) structure to become biologically active. To accomplish productive folding and transport However, about one-third of newly made pro- and to prevent off pathways, newly synthesized teins are cotranslationally transported to anoth- polypeptides interact with ribosome-associated er destination before folding, for example, to the chaperones that prevent inappropriate inter- endoplasmic reticulum (ER). The de novo fold- and intramolecular interactions and thus pro- ing and transport of proteins is problematic mote transport and/or folding into the native because hydrophobic residues of unfolded poly- state. Ribosome-associated chaperones are found peptide chains are accessible, which enhances in every cell but differ significantly among the the probability that the newly synthesized pro- different kingdoms of life with regard to their teins follow an unproductive off pathway leading structure and mechanism of action. Whereas to misfolding and aggregation or premature deg- prokaryotes have only one ribosome-associated radation (Deuerling and Bukau 2004; Hartl et al. chaperone, which is called trigger factor (TF), 2011; Balchin et al. 2016). Moreover, a large frac- eukaryotes use two different TF-unrelated tion of new proteins are cotranslationally modi- chaperone systems at the ribosome (Fig. 1), the fied, for example, by amino-terminal acetylation conserved heterodimeric nascent polypeptide-

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E. Deuerling et al.

Prokaryotes Eukaryotes

Yeast Mammals

30S Zuo1 40S MPP11 40S

50S 60S 60S

RAC RAC Trigger Ssz1 NAC Hsp70L1 NAC factor Ssb Hsp70

Figure 1. Ribosome-binding chaperones. The concept of ribosome-associated chaperones that assist de novo protein folding is conserved in prokaryotes and eukaryotes, albeit realized by different types of chaperones. The 30S or 40S ribosomal subunit is schematically drawn in dark gray and the 50S or 60S subunit in middle and light gray, indicating that the 50S/60S is sliced in half to illustrate the interior with the ribosomal tunnel. The nascent polypeptide (orange) attached to a transfer RNA (tRNA) in the P-site migrates through the tunnel and interacts with chaperones when it leaves the ribosome at the exit site. In prokaryotes (left), a single chaperone called trigger factor ([TF], red) binds transiently to the ribosome to welcome nascent polypeptides. In eukaryotes (right), two ribosome-associated chaperone systems exist: the heterodimeric nascent polypeptide-associated complex ([NAC], shown in pink and purple) and the ribosome-associated complex ([RAC], shown in yellow and light green), which consists of Ssz (light green) and Zuotin (Zuo, yellow) in yeast and the Zuo-homolog MPP11 (yellow) and its complex partner Hsp70L1 (light green) in mammals, respectively. Restricted to fungi, the Hsp70 Ssb (dark green) additionally binds to ribosomes and acts on nascent polypeptides, whereas in mammals RAC (MPP11/Hsp70L1) recruit a cytosolic Hsp70 (dark green) to bind to nascent polypeptides.

associated complex (NAC) and a specialized in eukaryotes (Steitz 2008; Jenner et al. 2012; Hsp70/Hsp40 dimeric chaperone system called Klinge et al. 2012). It consists of two subunits ribosome-associated complex (RAC). RAC acts in all organisms, a small subunit (30S in bacte- as acofactor foran additional ribosome-attached ria, 40S in eukaryotes) and a large subunit (50S Hsp70 partner in yeast (called Ssb) or a cytosolic in bacteria, 60S in eukaryotes). Four functional Hsp70 in higher eukaryotes (Deuerling and features are intrinsic to ribosomes (Fig. 2A): The Bukau 2004; Hartl et al. 2011; Preissler and ﬁrst feature is the decoding center, which en- Deuerling 2012). Recent analyses of the nascent sures selection of the correct aminoacyl-transfer interactome of these chaperones suggest that they RNA (tRNA) speciﬁed by the codon in the act on almost every new protein synthesized with mRNA. The decoding center lies in the small only a few exceptions (see below). A subset of subunit and recognizes the geometry of co- newly synthesized proteins bind to other cyto- don–anticodon base pairing and sterically dis- solic chaperones, for example, Hsp70 or Hsp60 criminates against mismatches (Schmeing and family members, later during synthesis or after Ramakrishnan 2009). The second feature repre- the release from ribosomes for further assistance sents the peptidyl-transferase center (PTC), the of their de novo folding program (for review, see active site of the ribosome where peptide bond Buskiewicz et al. 2004; Hartl et al. 2011; Preissler formation occurs. The PTC is located in a cleft and Deuerling 2012). within the subunit interface within the large ribosomal subunit. A third feature is the ribosomal tunnel inside the large subunit. With a length THE RIBOSOME AS PARTNER IN CRIME of 80–100 Å and a diameter of ∼10 Å at its The ribosome is a large ribonucleoprotein par- narrowest point and ∼20 Å at its widest point ticle of ∼2.4 MDa in bacteria and ∼4MDa it connects the PTC with the ribosome exit site

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Chaperone Interactions at the Ribosome

A B 30S/40S eL22

DC eL19 eL31 uL23 PTC uL29

eL39 H24 uL22

Tunnel 50S/60S Tunnel exit

Tunnel exit

Figure 2. Functional features of a ribosome. (A) Schematic depiction of a ribosome. The decoding center (DC) is located in the small ribosomal subunit (30S or 40S). Peptidyl-transferase center (PTC), ribosomal tunnel (tunnel), and the tunnel exit are located in the large ribosomal subunit (50S or 60S). A peptidyl-transfer RNA (tRNA) in the PTC with nascent chain (orange) is included to illustrate the path of a nascent chain. The constriction site within the ribosomal tunnel is indicated by the two arrowheads. (B) Top view on ribosome tunnel exit. Surface rendering of the yeast 60S subunit (gray) with ribosomal proteins implicated in cofactor binding around the tunnel exit (white circle) highlighted. eL19 (pale green), eL22 (green), uL22 (magenta), uL29 (marine), eL31 (red), eL39 (lime green). Helix 24 (H24) of the 25S ribosomal RNA (rRNA) is depicted in orange.

(Wilson and Beckmann 2011). The tunnel wall The tunnel exit has a diameter of ∼20 Å and is predominantly composed of ribosomal RNA is composed of rRNA and four conserved ribo- (rRNA) and only few ribosomal proteins. The somal proteins, uL22, uL23, uL24, and uL29 ribosomal proteins uL4 and uL22 contribute to (Fig. 2B). In addition, kingdom-speciﬁc ribo- the tunnel wall and form a 10 Å narrow con- somal proteins are also present at the exit site striction ∼30 Å from the PTC. The high content (e.g., eL19, eL31, eL39 in archea and eukaryotes; of rRNA gives the tunnel an overall electroneg- bL17 and bL32 only in bacteria). Some of these ative potential. Once thought of as a merely pas- ribosomal proteins have been shown to serve as sive conduit, the tunnel is now acknowledged to binding sites for ribosome-associated factors. In play an important role in protein biogenesis, for particular, uL23 associates with multiple na- example, in context of arrest sequences, which scent chain-processing factors, including TF, trigger PTC inactivation as well as forming a NAC, SRP, and Sec61, and was therefore dubbed secluded environment in which ﬁrst folding the “universal ribosome docking site” (Kramer events of the nascent polypeptide take place, et al. 2002; Pool et al. 2002; Wegrzyn et al. 2006). mostly the formation of α-helical elements (Wil- From a multitude of cross-linking and cryo- son and Beckmann 2011). The factor-binding electron microscopy (cryo-EM) studies, howev- platform centered around the tunnel exit repre- er, a more complex picture emerged, in which sents the fourth functional feature of the ribo- additional ribosomal proteins, such as eL19, some (Fig. 2B). It comprises several ribosomal eL22, uL22, uL29, eL31, and eL39 also represent proteins and rRNA elements and constitutes the crucial contact sites for ribosome-associated fac- binding sites for various ribosome-associated tors (Pool et al. 2002; Peisker et al. 2008; Pole- factors that act on nascent polypeptides includ- voda et al. 2008; Pech et al. 2010; Leidig et al. ing chaperones, processing enzymes (such as 2013; Zhang et al. 2014; Gumiero et al. 2016; Lee N-acetyltransferases [NATs]) and targeting fac- et al. 2016). In addition, ribosome-associated tors (e.g., signal recognition particle [SRP]). factors also contact rRNA elements (Leidig

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et al. 2013; Zhang et al. 2014; Gumiero et al. different conformations and contacts with the 2016; Lee et al. 2016). These interactions are ribosome have been suggested for the ATP- typically based on electrostatic interactions be- bound open and ADP-bound closed conforma- tween positively charged stretches within the as- tion (Gumiero et al. 2016). Clearly, we are only at sociated factors and negatively charged RNA. In the beginning of a detailed mechanistic under- general, ribosome association of individual fac- standing and appreciation of the intricate dy- tors is highly dynamic and in cases in which namic processes taking place at the ribosome factors compete for the same or close nearby exit site and within the ribosomal tunnel. binding site(s), binding of factors is often mutu- ally exclusive. Notably, the order in which factors associate with the ribosome and bind the STRUCTURES, FUNCTIONS, AND nascent chain is primarily determined by the MECHANISMS OF ACTION OF RIBOSOME- chemical attributes of the emerging nascent ASSOCIATED CHAPERONES chain itself. Bacterial Trigger Factor It is likely that several factors can adopt different conformations on the ribosome and have TF is a highly abundant chaperone found in more than one binding site on the ribosome de- bacteria and chloroplasts but not in the cytosol pending on their functional state. For example, of eukaryotes. It associates via a ribosome-bind- for the ribosome-attached Hsp70 Ssb in yeast, ing motif in its ND (Fig. 3) transiently in a 1:1

AC Tall ribosome binding Head PPlase arm1 arm2 1 118 149 250360 432

-GFRxGxxP- 43 50

B PPlase

arm1 arm2

uL23

Ribosome-binding motif

Figure 3. Escherichia coli trigger factor (TF). (A) Schematic representation of the linear domain organization of TF. The ribosome-binding domain (“tail”) with the ribosome-binding motif (residues 43–50) is shown in red, the PPIase “head” in green and “arm” 1 and “arm” 2 in light blue and dark blue, respectively. (B) TF adopts an extended three-dimensional fold. (Left) Ribbon diagram of the TF fold. Color code is similar to A. In positions of the ribosome-binding motif (residues 43–50), the arms and the PPIase are indicated. (Right) Surface charge distribution of TF in the same orientation as in the ribbon diagram. Positively and negatively charged residues are shown in blue and red, respectively. (C) Structural model of TF bound to the ribosome (different shades of gray as in Fig. 1). The main contact between TF (colors as in A) involves the ribosome-binding motif in the N domain and the ribosomal protein uL23 (light gray). The nascent polypeptide (yellow) migrates into the TF cradle during synthesis. All TF structures were prepared using PyMOL (DeLano Scientiﬁc LLC) based on Ferbitz et al. (2004).

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Chaperone Interactions at the Ribosome

ratio with ribosomes mainly via interaction with substrate-binding sites distributed along TFs’ the ribosomal protein uL23 at the tunnel exit inner surface (Fig. 3B) with a variable order of (Kramer et al. 2002; Ferbitz et al. 2004). First binding and occupancy. The binding sites are evidence that TF acts as a chaperone for nascent composed of nonpolar residues that can form polypeptides was provided by experiments numerous hydrophobic pockets to bind to hy- showing that combined loss of TF and the drophobic peptide stretches of 6–10 residues in Hsp70 chaperone DnaK provoked synergistic substrate proteins. Additionally, polar residues defects in de novo protein folding resulting in proximate to the hydrophobic-binding sites can global protein aggregation and decreased viabil- be used to form hydrogen bonds with the sub- ity of Escherichia coli cells (Deuerling et al. 1999; strate probably to enhance affinity and navigate Teter et al. 1999). binding. Importantly, these binding sites show a TF has a unique 3D conformation. It is an flexible local architecture and the engagement of extended dragon-like structure with a central individual residues within the binding sites is body including two protruding arms, a head, variable depending on the substrate segment and a tail region (Fig. 3). Bound to the ribosome bound to it. This high degree of plasticity of as monomer by its amino-terminal tail, TF leans TFs’ binding surfaces explains how TF can serve over the ribosomal exit tunnel thereby exposing such a promiscuous and large pool of nascent its large interior surface speckled with multiple substrates (Gamerdinger and Deuerling 2014; hydrophobic patches toward the exiting nascent Saio et al. 2014). Using up all of its binding sites, chain (Fig. 3B,C; Ferbitz et al. 2004). Thus, TF is TF can directly bind up to 50 amino-acid resi- ideally positioned to capture emerging chains. dues of a substrate. The hydrophobic peptide Based on the crystal structure, TF can accom- stretches bound by TF are separated by linker modate entire protein domains or even small regions in between that remain unbound and proteins (up to a length of 130 aa) in its central may even loop outward of the central cavity. cavity. Ribosome-bound TF can prevent prema- Perhaps, multiple TF molecules can bind simul- ture and incorrect folding of proteins during taneously to a nascent substrate, which enables synthesis. For example, TF retards cotransla- TF to retain also large polypeptides in an unfold- tional folding of recombinantly expressed firefly ed state and protect them from aggregation by luciferase in E. coli cells, thereby enhancing the shielding their exposed hydrophobic regions. total yield of active luciferase (Agashe et al. 2004; This interaction mode of TF may even exert Kaiser et al. 2006). More recent data suggest that the force driven by the binding energy to unfold TF can reshape and improve the folding pathway misfolded peptide segments of low thermody- of a protein by protecting partially folded inter- namic stability. mediates (Singhal et al. 2015; Wruck et al. 2018). More than 300 different aggregation-prone Even more intriguing, TF can reverse premature protein species were found in TF- and DnaK- folding by facilitating unfolding of preformed deficient E. coli cells. The identified proteins structures in nascent polypeptides, which allows are involved in many different cellular processes, the nascent peptide to reenter the productive range in size from 16 kDa to 140 kDa, and are folding path (Hoffmann et al. 2012; Mashaghi specifically enriched for large (>40 kDa) multi- et al. 2013). domain proteins (Deuerling et al. 2003). Selec- A more detailed understanding of the TF tive ribosome profiling (SeRP) provided the first mechanism of action was provided by a recent global analysis of the nascent interactome of TF study of Saio and colleagues using sophisticated (Oh et al. 2011). This technique combines affin- nuclear magnetic resonance (NMR) techniques ity purification of ribosome–TF complexes and (Gamerdinger and Deuerling 2014; Saio et al. subsequent identification of the mRNA segment 2014). These investigators determined the struc- that is being read by TF-bound ribosomes ture and dynamics of purified TF interacting (Becker et al. 2013). This elegant approach with unfolded model substrates in solution. TF revealed new fundamental features of the co- forms a binding scaffold with up to four distinct translational activity of TF. TF recruitment to

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translating ribosomes is delayed in vivo until also available for the carboxyl terminus of nascent peptides reach a length of ∼100 aa. As- αNAC, which forms a compact three-helix-bun- suming that 30–40 aa of the nascent chains are dle motif characteristic for ubiquitin-associated buried in the ribosomal tunnel, TF must bind to domains (UBAs) (Fig. 4B) (Spreter et al. 2005). emerging peptides only once they have exposed The remaining parts of NAC including the NDs at least 60–70 aa outside the ribosome. This ini- of both subunits and the carboxy-terminal do- tial exclusion of TF, which only correlates with main of βNAC have not been structurally re- the length of the polypeptides, provides a time solved yet, and many regions in these domains window to allow processing enzymes to access are predicted to be intrinsically disordered, sug- nascent proteins (Oh et al. 2011). These enzymes gesting that overall NAC shows high flexibility are required, for example, to remove the formyl- (Pech et al. 2010; Martin et al. 2018). moiety and the initiator methionine from the NAC is abundantly expressed in equimolar amino termini of nascent polypeptide chains. concentration relative to ribosomes and revers- The second important finding from the ribo- ibly binds in a 1:1 stoichiometry to ribosomes some profiling study is that TF interacts with in close proximity to the ribosomal tunnel exit all newly synthesized proteins, except for those (Raue et al. 2007; Preissler and Deuerling 2012). that are inserted into the cytoplasmic membrane Cross-linking data suggest that both subunits via the SRP targeting pathway. In contrast, na- can interact with nascent chains (Wiedmann scent outer membrane β-barrel proteins (Omps) et al. 1994), while specifically the amino-termi- were among the strongest TF interactors during nal region of βNAC is critical for ribosome bind- their synthesis on ribosomes. This suggests that ing (Wegrzyn et al. 2006; Pech et al. 2010). Stud- the chaperone activity of E. coli TF is particularly ies in yeast showed that deletion of the first 11 important for keeping Omps in a translocation- amino-terminal residues or mutation of a con- competent conformation, so that they can be served positively charged motif in the center efficiently exported by the Sec machinery (Oh (RRKxxKK) abolishes ribosome binding, sug- et al. 2011). It should be mentioned that this gesting that the amino-terminal part of βNAC finding is in agreement with pioneering studies makes the main ribosomal contact (Wegrzyn by Wickner and coworkers, who in 1987 initial- et al. 2006; Pech et al. 2010). Cross-linking ly identified E. coli TF in a reconstituted in data suggest that βNAC contacts the ribosome vitro translocation experiment, as a cytosolic via the ribosomal protein uL23 (Wegrzyn et al. component that maintains proOmpA in a 2006); however, other studies also suggest an transport-competent conformation for delivery interaction with eL31 (Pech et al. 2010; Zhang into inside-out membrane vesicles (Crooke and et al. 2012; Nyathi and Pool 2015). Both ribo- Wickner 1987). somal proteins are located next to the nascent peptide tunnel exit but on opposite sides (Fig. 4C), indicating some flexibility of NAC Eukaryotic Ribosome-Associated Systems on ribosomes. In addition, also the α-subunit The Nascent Polypeptide-Associated may contribute to ribosome binding as a recent α Complex study showed a cross-link between NAC and the ribosomal protein uL29, a neighboring The NAC is an evolutionarily conserved eukary- protein of uL23 (Fig. 4C) (Nyathi and Pool otic heterodimeric protein complex composed 2015). However, apart from these cross-linking of a α- and β-subunit, referred to as αNAC and data no further structural information is avail- βNAC (Fig. 4A) (Wiedmann et al. 1994). Both able yet and the exact positioning and structure subunits contain a homologous NAC domain, of NAC at the ribosomal tunnel exit remains which dimerize by forming a β-barrel-like obscure. structure with a hydrophobic core (Fig. 4A,B) The abundance and the position at the ribo- (Liu et al. 2010; Wang et al. 2010). Besides the somal tunnel exit indicate a central role for NAC dimerization domain, structural information is in guarding de novo protein synthesis. The

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Chaperone Interactions at the Ribosome

AC αNAC NAC UBA 1 84 132 176 215 40S βNAC NAC 1 53 110 162 -RRKKK- 27 31 B βNAC UBA domain uL23

αNAC

uL29 eL31

60S Tunnel exit N C

NAC domain

Figure 4. The nascent polypeptide-associated complex (NAC) in eukaryotes. (A) Schematic representation of the linear domain organization of human αNAC and βNAC. Both subunits contain a homologous NAC dimerization domain. A conserved positively charged ribosome-binding motif (RRKKK) is located in the amino-terminal domain of βNAC. A conserved ubiquitin-associated domain (UBA) is located at the very carboxyl terminus of αNAC. (B) Crystal structure of the human NAC dimerization domain, which forms a compact β-barrel-like structure (blue, βNAC; violet, αNAC, PDB: 3MCB). The structure of the UBA domain (from archaeal NAC, PDB: 1TR8) consists of a typical three-helix-bundle. Gray dashed lines indicate parts of NAC that are not structurally resolved yet. (C) Surface rendering of yeast 60S (gray) and 40S (wheat) subunits with potential major contact points of NAC near the tunnel exit revealed by cross-linking experiments. βNAC cross-links to both uL23 and eL31, whereas αNAC cross-links to uL29.

deletion of NAC leads to embryonic lethality NAC was found to be essential, indicating that in worms, flies, and mice, demonstrating a NAC functions as a regulatory device blocking a fundamental role of NAC in the protein homeo- high-affinity Sec61-binding site on ribosomes stasis network (Deng and Behringer 1995; Mar- near the tunnel exit. This hypothesis is based kesich et al. 2000; Bloss et al. 2003). Despite its on the fact that ribosomes per se show a very essential role, the in vivo function of NAC high intrinsic affinity to the Sec61 complex remained enigmatic for a long time and only in the low nanomolar range independent of recently important functional insights were ob- whether or not a signal sequence is exposed tained in Caenorhabditis elegans showing a (Borgese et al. 1974; Jungnickel and Rapoport primary function of NAC in regulating the 1995). Thus, to maintain ER targeting specificity cotranslational protein transport to the ER the binding of ribosomes to Sec61 must be neg- (Gamerdinger et al. 2015). In the absence of atively regulated by NAC. This is in agreement NAC, translating ribosomes unspecifically asso- with a previous in vitro study showing that pu- ciate with the Sec61 translocon in the ER mem- rified NAC prevents unspecific binding of ribo- brane leading to the mislocalization of nascent somes to ER-derived membranes (Moller et al. substrates to the ER. To prevent incorrect ribo- 1998). The activity of NAC opposes that of the some–Sec61 interactions, ribosome binding of SRP, which promotes the binding of correct,

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signal sequence-containing, ribosome-nascent growth of yeast cells treated with the proline an- chain complexes (RNCs) to the ER translocon alog azetidine-2-carboxylic acid (AZC), which (Keenan et al. 2001). This antagonistic “sort and is known to corrupt the folding of newly syn- countersort” interplay between NAC and SRP is thesized proteins (Duttler et al. 2013). NAC essential to enhance accuracy and efficiency of deletion also exacerbates widespread protein the cellular protein-sorting machinery (Gamer- aggregation in yeast cells lacking the major, dinger et al. 2015). The pivotal physiological ATP-driven cotranslational chaperone system, role of NAC in ER protein transport is under- the RAC–Ssb system (Koplin et al. 2010). Alto- scored by the fact that NAC deficiency leads to gether, these data support a role for NAC as an strong induction of the unfolded protein re- early acting molecular chaperone assisting the sponse (UPR) in the ER and accompanying in- cotranslational folding of nascent chains. The duction of cell apoptosis, as shown in C. elegans, principle that ATP-independent ribosome-as- zebrafish, and human cells (Hotokezaka et al. sociated chaperones have a crucial function in 2009; Arsenovic et al. 2012; Murayama et al. the folding of many newly synthesized proteins 2015). Moreover, in addition to controlling the is well established for TF in bacteria, as outlined inherent Sec61–ribosome-binding affinity, NAC above. TF protects nascent chains against pre- may be also important to regulate the binding mature aggregation and degradation by using specificity of SRP to translating ribosomes. A several flexible binding sites to shield hydropho- previous in vitro study indicated that NAC is bic peptide stretches in substrate proteins. How- required to prevent SRP to bind to signal se- ever, whether NAC acts similar to the holdase quenceless RNCs, indicating that NAC and chaperone TF is speculative. Apart from cross- SRP use overlapping binding sites on ribosomes linking data suggesting that both NAC subunits and NAC inhibits the low-affinity binding of interact with nascent chains, little is known SRP to nonsecretory RNCs (Wiedmann et al. about NAC’s substrate-binding specificity and 1994). This finding was partially reproduced in even the particular substrate-binding site(s) of vivo in yeast, showing that NAC modulates SRP- αNAC and βNAC have not been mapped so binding specificity to some degree (del Alamo far. A recent cross-link-mass spectrometry study et al. 2011). However, the NAC–SRP interplay indicated that NAC predominantly interacts remains obscure and is controversially dis- with substrates via the flexible amino-terminal cussed, as other in vitro studies did not find regions of αNAC and βNAC; however, whether a evidence for an altered SRP-binding specificity crucial chaperone domain is located in these do- (Neuhof et al. 1998; Raden and Gilmore mains is yet unknown (Martin et al. 2018). Stud- 1998), and, most importantly, because overall ies in yeast showed that NAC can associate with SRP-directed ER membrane targeting of RNCs practically every ribosome-attached nascent seems not to be affected in vivo in the absence of polypeptide of a cell indicating that NAC may NAC (del Alamo et al. 2011; Gamerdinger et al. serve a very large pool of nascent substrates (del 2015). Alamo et al. 2011). In contrast to other organ- In addition to its function as a negative reg- isms, yeast cells express two different βNAC sub- ulator of ER protein transport, several lines of units, βNAC and β0NAC, the latter expressed in evidence also suggest a function of NAC as an lower amounts (Ott et al. 2015). Interestingly, ATP-independent molecular chaperone. Cross- the cotranslational substrate pool greatly dif- linking data indicate a direct binding of NAC to fered between the two NAC species that exist nascent chains and NAC deletion in yeast and in yeast. NAC containing β0NAC preferentially human cells leads to an increased ubiquitylation associates with proteins showing high intrinsic of nascent polypeptides suggesting that NAC disorder and lower hydrophobicity. Conversely, directly binds to nascent substrates to protect NAC containing the β-subunit binds to longer them from premature degradation (Wiedmann polypeptides with high hydrophobicity and low- et al. 1994; Duttler et al. 2013; Wang et al. 2013). er intrinsic disorder (del Alamo et al. 2011). Furthermore, NAC is required to promote These differences indicate that each NAC sub-

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Chaperone Interactions at the Ribosome

unit recognizes specific physicochemical prop- The Ribosome-Associated Complex erties of the nascent polypeptide. However, β0NAC is only present in yeast, suggesting that Eukaryotes feature a second conserved ribo- its specific function is either not required or some-associated chaperone system that is in- adopted by canonical NAC or other proteins in volved in initial protein folding, targeting, and higher eukaryotes. prevention of aggregation of newly synthe- Further evidence also suggests a function for sized proteins, the RAC. RAC acts together NAC in protein transport to mitochondria. with a ribosome-bound Hsp70 (heat-shock 70 Knockdown of NAC induces the mitochondri- kDa protein) in yeast (Ssb) or recruits cytosolic al UPR in C. elegans and causes mitochondrial Hsp70 to nascent polypeptides in mammals dysfunction in human cells (Hotokezaka et al. (Fig. 1B,C) (Preissler and Deuerling 2012; 2009; Gamerdinger et al. 2015). Moreover, in Zhang et al. 2017). RAC is a stable heterodimeric yeast, NAC was found to enhance the efficiency complex of an Hsp40 protein (Zuo1 in yeast, of protein import to mitochondria (George et al. ZRF1/MPP11 in humans) and a degenerated 1998; Fünfschilling and Rospert 1999). In this ATPase-inactive Hsp70 protein (Ssz1 in yeast, respect, NAC promotes the interaction of ribo- Hsp70L1 in humans) (Fig. 5A,B) (Gautschi somes with the mitochondrial surface, suggest- et al. 2001, 2002; Huang et al. 2005; Conz et al. ing that NAC stimulates import of mitochondri- 2007). Most of our knowledge of the function al proteins in a cotranslational manner (George and structure of the RAC–Hsp70 system is de- et al. 2002). A recent study in yeast showed that rived from studies in the baker’s yeast Saccharo- cotranslational protein transport to mitochon- myces cerevisiae. RAC stimulates the ATPase dria is more widespread than previously thought activity of Ssb in yeast and thereby enhances (Williams et al. 2014). The targeting process of the affinity of this Hsp70 for unfolded polypep- RNCs exposing a mitochondrial targeting se- tides ultimately assisting de novo protein folding quence (MTS) to the translocase (TOM com- (Preissler and Deuerling 2012; Zhang et al. plex) of the organelle is not yet established. 2017). RAC itself seems not to directly contact However, NAC could play a major role therein the nascent chain (Gautschi et al. 2002; Conz as it binds to the mitochondrial outer mem- et al. 2007), but nonetheless it plays an impor- brane protein OM14 in yeast (Lesnik et al. tant role in coordinating the substrate-binding 2014). Through this interaction, translating ri- specificity of Ssb in yeast (Koplin et al. 2010; bosomes get localized to mitochondria and both Willmund et al. 2013; Döring et al. 2017). A NAC and OM14 are required to enhance pro- functional cooperation between components of tein import efficiency. Consistent with a mito- the yeast Hsp40/Hsp70–chaperone triad was chondrial targeting function, β0NAC was found first revealed by genetic analyses. Cells lacking to associate preferentially with ribosomes trans- either Ssb or RAC or both display a similar phe- lating mitochondrial precursors in yeast (del notype, which includes sensitivity to high salt Alamo et al. 2011). Moreover, mitochondrial concentrations, low temperature, and transla- proteins in particular get mistargeted and mis- tion inhibitory drugs such as aminoglycosides localized to the ER on NAC depletion in (Nelson et al. 1992; Yan et al. 1998; Gautschi C. elegans (Gamerdinger et al. 2015). In sum, et al. 2002; Hundley et al. 2002). The first hint these findings support a function of NAC in to a role of the RAC–Ssb system in folding of cotranslational protein transport to mitochon- nascent chains at the ribosome came from dria. However, the question arises how NAC, cross-linking experiments establishing a RAC- which broadly binds to RNCs, can exert dis- dependent interaction of Ssb with short nascent criminative targeting activity toward RNCs, ex- chains (Pfund et al. 1998; Gautschi et al. 2002; posing an MTS. Moreover, OM14 is not con- Hundley et al. 2002). An involvement of RAC– served and a potential receptor for NAC on Ssb in de novo protein folding was further cor- mitochondria in higher organisms has not roborated by the fact that the aminoglycoside been identified yet. sensitivity of racΔ ssbΔ yeast cells can be partially

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Figure 5. The ribosome-associated Hsp70/40 system in eukaryotes. (A) The yeast ribosome-associated chaperone system consists of the Hsp40 Zuo1, and the Hsp70s Ssb and Ssz1. Zuo1 and Ssz1 form the stable heterodimeric ribosome-associated complex (RAC). Zuo1 binds to the Ssz1 nucleotide-binding (NBD) and substrate-binding domain β (SBDβ) via its amino- o:10.1101/cshperspect.a033977 doi: terminal domain (ND). The J domain (JD) of Zuo1 is required for stimulation of the ATPase activity of its Hsp70 partner Ssb. The Zou1 homology domain (ZHD), the highly charged middle domain (MD), and the four-helix bundle (4HB) are involved in ribosome binding. Ssz1 and Ssb contain an amino-terminal NBD and a carboxy-terminal SBD. Ssz1 contains an incomplete SBD, which consists of only the SBDβ moiety, whereas the SBD of Ssb is complete and consists of SBDα and SBDβ. Autonomous ribosome binding of Ssb is mostly mediated via a positively charged stretch at the carboxyl terminus. RAC is conserved in mammals while Ssb homologs are absent (and a soluble Hsp70 is used instead). (B) The human RAC complex is formed by the Hsp40 MPP11 and Hsp70L1. MPP11 contains two SANT domains at its carboxyl terminus. (C) Surface rendering of yeast 60S (gray) and 40S (wheat) subunits with major contact points with Zuo1 highlighted. The dotted empty outline coarsely traces the shape of Zuo1. Exit tunnel (white circle), uL22 (magenta), uL31 (red), helix 24 (H24) of the 25S ribosomal RNA (rRNA) in 60S subunit (orange), and helix 44 (H44) of expansion segment 12 of 18S rRNA in 40S subunit (cyan). S. cerevisiae, Saccharomyces cerevisiae. Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press

Chaperone Interactions at the Ribosome

suppressed by expression of the prokaryotic ri- H24 and the ZHD to the Zuo1 J domain, which bosome-associated chaperone TF (Rauch et al. may influence J domain positioning with respect 2005). In addition, cells devoid of Ssb showcase to Ssb and hence ultimately regulate Ssb activity an increased aggregation of newly synthesized at the tunnel exit (Lee et al. 2016; Zhang et al. proteins, as well as of ribosomal proteins and 2017). Moreover, Zuo1 interacts via a positively ribosome biogenesis factors (Koplin et al. 2010; charged surface with helix 44 (H44) of extension Willmund et al. 2013). These findings indicate segment 12 (ES12) of 18S rRNA in the 40S sub- that a loss of Ssb not only negatively impacts on unit (Zhang et al. 2014; Lee et al. 2016). Because folding of cytosolic proteins but also on ribo- the extended H44 forms the core of the decoding some biogenesis. Interestingly, the RAC–Hsp70 center (DC) and mutations within H44 affect system is also important for maintaining trans- translational fidelity (Lee et al. 2016), binding lational fidelity (Rakwalska and Rospert 2004; of Zuo1 to the tip of H44 might explain how Muldoon-Jacobs and Dinman 2006; Lee et al. RAC influences translational fidelity (Lee et al. 2016; Zhang et al. 2017). 2016; Zhang et al. 2017). Recent structural work significantly expand- An unusual attribute of RAC is the stable ed our understanding of the fungal RAC–Ssb interaction of the Hsp40 Zuo1 and the degener- system on the ribosome (Leidig et al. 2013; ated Hsp70 Ssz1, because Hsp40–Hsp70 inter- Zhang et al. 2014, 2017; Lee et al. 2016). Ribo- actions are normally transient (Mayer and Kityk some binding of the RAC complex is mediated 2015). First biochemical insights into the sta- solely by the Zuo1 subunit, which contacts the ble interaction of the two proteins came from ribosomal protein eL31 close to the tunnel exit hydrogen–deuterium exchange experiments, via a conserved charged region (Yan et al. 1998; which showed that the Zuo1 amino-terminal Peisker et al. 2008). Moreover, recent cryo-EM domain (ND) binds stably to Ssz1, making di- studies unveiled a much more intricate interac- rect contacts with both the substrate-binding tion between Zuo1 and the ribosome. Zuo1 not domain (SBD) and the nucleotide-binding do- only contacts the 60S subunit but also the 40S main (NBD) of Ssz1 (Fiaux et al. 2010). The subunit and spans ∼190 Å across the subunits recent crystal structure of full-length Ssz1 in (Fig. 5A,C) (Zhang et al. 2014; Lee et al. 2016). complex with the Zuo1 ND (both from the ther- While the central Zuo1 homology domain mophilic ascomycete Chaetomium thermophi- (ZHD) binds the 60S subunit (Leidig et al. lum) fully confirmed these findings and further 2013; Zhang et al. 2014; Kaschner et al. 2015; revealed unique features of the interaction of the Lee et al. 2016), a four-helix bundle (4HB) at two proteins (Weyer et al. 2017; Zhang et al. the carboxyl terminus of Zuo1 binds to the 40S 2017). In contrast to canonical Hsp70s, the link- subunit, and the middle domain (MD) forms a er between NBD and SBD is longer in Ssz1, and hinge between the two domains (Zhang et al. is detached from ATP-bound NBD allowing 2014; Lee et al. 2016). Zuo1 ND to bind this site in trans instead. In The unique binding mode of RAC to trans- addition, parts of the Zuo1 ND complement the lating ribosomes has several mechanistic impli- β-sandwich of Ssz1 SBDβ thereby mimicking cations. A communication between the ribo- SBD β of canonical ADP-bound Hsp70s (despite somal tunnel and chaperone activity at the having ATP bound). Overall, the conformation tunnel exit has been anticipated given that of Ssz1 in the RAC heterodimer looks like a Zuo1 contacts helix 24 (H24) of the 25S rRNA hybrid between ADP- and ATP-bound Hsp70 in the 60S subunit, and H24 in turn is in contact (Weyer et al. 2017). with ribosomal protein uL22, which extends into Ssz1 represents in several aspects an atypical the ribosomal tunnel and makes contact with Hsp70: First, it binds ATP but is not able to nascent chain segments at the constriction site. hydrolyze it (at least at a detectable rate), and Conceivably, the nature of the nascent chain in ATP binding is not strictly required for its func- the tunnel is sensed at the constriction site by tion (Huang et al. 2005; Conz et al. 2007). Sec- uL22 and the information might be relayed via ond, it lacks the lid domain of the SBD and no

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binding to a substrate could be detected (Hund- somes (Pfund et al. 1998). In contrast, Ssb bind- ley et al. 2002; Huang et al. 2005). An Ssz1 trun- ing to ribosomes exposing a nascent chain cation variant lacking the entire SBD fully com- (RNCs) is resistant to high salt concentrations, plements growth defects of an ssz1Δ strain presumably because of hydrophobic Ssb–na- (Hundley et al. 2002; Conz et al. 2007). Zuo1 scent chain interactions (Powers and Walter can only efficiently stimulate ATP hydrolysis by 1996; Pfund et al. 1998; Raue et al. 2007). A sec- Ssb in the presence of Ssz1 (Huang et al. 2005). ond positively charged region within the Ssb Consequently, it was proposed that Ssz1’spre- SBDβ in addition contributes to ribosome bind- dominant function is to facilitate Zuo1’s ability ing (Hanebuth et al. 2016). Cross-linking exper- to function as a J protein partner of Ssb on the iments revealed that Ssb contacts ribosomal pro- ribosome (Huang et al. 2005). It was also spec- teins uL29, eL39, and eL19 in close proximity of ulated that Ssz1 might fulfill regulatory func- the tunnel exit (Gumiero et al. 2016). Together tions by introducing structural rearrangements with the crystal structure of full-length C. ther- within in the Zuo1 J domain, which might mophilum Ssb (in the ATP-bound state), a pic- strengthen the contact to Ssb (Fiaux et al. ture emerges in which an exact positioning of Ssb 2010), and that Ssz1 might play a role in re- on the ribosome close to the exit tunnel is critical cruitment of substrates by Ssb (Leidig et al. for its function (Gumiero et al. 2016; Hanebuth 2013). Regardless, despite the immense pro- et al. 2016). The positioning of Ssb on the ribo- gress on the structural site, the function of the some is modulated by RAC (as well as the nucle- unique RAC SBDβ conformation, as well as the otide-status of Ssb itself) indicating dynamic mechanistic role of Ssz1 remain hitherto largely changes of Ssb on the ribosome on ATP hydro- enigmatic. lysis (Gumiero et al. 2016). Intriguingly, in the In yeast, two Ssb homologs exist, Ssb1 and presence of RAC, autonomous ribosome bind- Ssb2, which differ only in four amino acids (here, ing of Ssb is not essential for protein folding, collectively referred to as Ssb). The structure of suggesting a RAC-mediated interaction of Ssb Ssb is similar to canonical Hsp70 proteins, ex- with RNCs (Gumiero et al. 2016; Hanebuth cept that it contains in addition a nuclear export et al. 2016). This is mostly consistent with the sequence (NES) at its carboxyl terminus, which situation in organisms other than fungi, in which likely facilitates shuttling between the nucleus no dedicated autonomously ribosome-anchored and cytosol (Shulga et al. 1999). As for canonical Hsp70 exists and RAC collaborates with a cyto- Hsp70s, the Ssb reaction cycle is driven by co- solic Hsp70 instead (Fig. 1; Nelson et al. 1992; chaperones (Preissler and Deuerling 2012; Gautschi et al. 2002). Mayer and Kityk 2015). ATP hydrolysis, which Two recent studies investigated the cotrans- results in tight substrate binding, is stimulated by lational substrates of Ssb on a global scale (Will- RAC (Gautschi et al. 2002; Hundley et al. 2002), mund et al. 2013; Döring et al. 2017). In the first and nucleotide exchange factors for Ssb are Sse1, study by Willmund and colleagues, ribosome– Sln1, and Fes1 (Peisker et al. 2010). The highly Ssb complexes were isolated and the ribosome- abundant Ssb interacts independent of RAC with associated mRNA analyzed using genome-wide ribosomes, and in wild-type cells ∼50% of Ssb is microarrays. This study revealed that the na- found associated with ribosomes (Nelson et al. scent chain substrate pool of Ssb is very broad, 1992; Yan et al. 1998; Peisker et al. 2010). A pos- encompassing ∼80% of cytosolic and nuclear itively charged region close to the end of the proteins. General features of the Ssb-bound pro- carboxy-terminal SBDα is essential for ribosome teins were the presence of larger domains and a binding (Gumiero et al. 2016; Hanebuth et al. generally large size. Ssb substrates were also en- 2016). Likely, this region mediates ribosome as- riched for subunits of oligomeric complexes sociation of Ssb via electrostatic interactions with (e.g., TRiC/CCT, proteasome, and ribosomal expansion segment ES24 and/or ES41 within the particle), which in most cases do not represent 25S rRNA (Gumiero et al. 2016), consistent with large proteins, but usually interact with several a salt-sensitive binding of Ssb to vacant ribo- other subunits within the complex. Further fea-

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Chaperone Interactions at the Ribosome

tures common to most Ssb substrates were a way before docking to the translocon, at least for general low translation rate, and an enrichment a subset of SRP-targeted nascent chains, Ssb en- of β-sheets, and short linear hydrophobic ele- gagement before SRP binding has been observed ments. Together, these data suggest that Ssb as- (Döring et al. 2017). Whether this implies a sists cotranslational folding of larger proteins handover of nascent chains from Ssb–RAC to harboring complicated structures or that require SRP and/or the existence of an alternative tar- binding partners for their stability (Willmund geting route to the ER membrane is currently et al. 2013). unknown (Döring et al. 2017). In the second analysis, SeRP (Becker et al. An influence of RAC on the substrate speci- 2013) was used to analyze Ssb interaction with ficity of Ssb was observed in both studies (Will- nascent chains on a proteome-wide scale at mund et al. 2013; Döring et al. 2017). The ab- near-codon resolution (Döring et al. 2017). sence of RAC severely impaired Ssb binding to Consistent with Willmund et al. (2013), Ssb emerging recognition motifs and delayed RNC bound to ∼80% of cytosolic and nuclear proteins engagement of Ssb (Döring et al. 2017). In in the SeRP study. Furthermore, and in dissent addition, deletion of RAC was found to relax with Willmund et al. (2013), ∼80% of nascent the specificity of Ssb (Willmund et al. 2013). mitochondrial proteins and more than 40% of Intriguingly, Ssb binding to RNCs coincides ER-targeted proteins were found associated with with an increased translation speed, which Ssb, suggesting a function of Ssb in targeting/ can be attributed to both mRNA features as translocation of these proteins (Döring et al. well as nascent chain features (Döring et al. 2017). Because Ssb was found to bind nascent 2017). It has been speculated that faster trans- mitochondrial proteins preferentially at lengths lation could reduce the number of ribosomes of 100 residues, it has been speculated that Ssb required to maintain protein synthesis and might increase the targeting efficiency of these may therefore represent a strategy to economize proteins by preventing premature folding and protein synthesis (Döring et al. 2017). Either misfolding (Döring et al. 2017). Such a scenario way, this finding highlights the close coordina- is supported by the fact that a subset of nuclear- tion of protein synthesis and cotranslational encoded mitochondrial proteins aggregated in protein folding. the absence of Ssb (Willmund et al. 2013). The Whereas Ssb is unique to fungi, homologs of SeRP study further showed that Ssb binds posi- RAC are present in mammals as well (Hundley tively charged, degenerate sequences close to the et al. 2005; Otto et al. 2005). This indicates that ribosomal surface, when the substrate recogni- the presence of Hsp70/40 chaperones on ribo- tion motif extends ∼5 aa from the tunnel exit, somes is common in the eukaryotic world. Zuo1 whereby disordered regions seem to be disfa- homologs in mammals (MPP11) contain two vored. Ssb nascent chain binding close to the additional SANT domains at their carboxyl ter- tunnel exit is in good agreement with a wealth minus (Hundley et al. 2005; Otto et al. 2005; of previous biochemical and structural data Chen et al. 2014). SANT domains are normally (Pfund et al. 1998; Gautschi et al. 2002; Hundley involved in DNA binding and chromatin re- et al. 2002; Gumiero et al. 2016). Notably, Ssb modeling (Boyer et al. 2004), their exact role in engages most substrates by multiple binding-re- MPP11 however is currently unknown. Knock- lease cycles (Döring et al. 2017). As Ssb binds down of human MPP11 in HeLa cells results in preferentially segments that will be surface ex- growth defects and sensitivity toward drugs sim- posed or form the hydrophobic core of the fold- ilar to what was observed for yeast cells lacking ed protein, Ssb likely assists domain-wise folding RAC (Jaiswal et al. 2011), suggesting that RAC by retarding premature/unproductive folding fulfills similar functions in yeast as well as in (Döring et al. 2017). metazoans. However, because higher eukaryotes Challenging the common view that SRP is lack a dedicated ribosome-anchored Hsp70-like the first and only cytosolic interactor of nascent Ssb, it was suggested that cytosolic Hsp70s act as chains of the cotranslational translocation path- functional partners for RAC in higher eukary-

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otes (Jaiswal et al. 2011). Interestingly, although binding, targeting of RNC to the membrane via Zuo homologs are found in all eukaryotic spe- SRP–SRP receptor (FtsY) assembly, and removal cies in which the whole genome is sequenced, of SRP from RNCs exposing nascent chains not all of them also reveal an Ssz-like Hsp70 exceeding a critical length that compromises (e.g., there is no Ssz homolog found in plants), cotranslational translocation (Buskiewicz et al. suggesting that the RAC system may have a more 2004; Bornemann et al. 2014; Ariosa et al. versatile setting than the yeast and mammalian 2015). Together, these activities enhance the fi- versions characterized so far. delity of substrate selection by SRP and promote the efficiency of membrane protein transport in the cell. In contrast to SRP, TF seems not to INTERPLAY WITH OTHER RIBOSOME- modulate directly PDF and MAP binding to ASSOCIATED FACTORS: SRP, PDF, MAP, translating ribosomes, indicating that these en- AND NAT zymes can process their nascent substrates be- The ribosome-associated chaperone systems fore or in parallel with TF binding (Bornemann presented in this review are integral and essential et al. 2014). parts of a greater cotranslational proteostasis The regulatory function of TF in cotransla- machinery that controls the quality and locali- tional protein transport partially resembles that zation of newly synthesized proteins. Numerous of NAC in eukaryotes. As outlined above, NAC other cytosolic protein biogenesis factors, in- is critical for accurate substrate selection by the cluding peptide deformylase (PDF), methionine Sec61 translocon, but also modulates the bind- aminopeptidases (MAPs), NATs, and the SRP ing specificity of SRP (del Alamo et al. 2011; also bind in close proximity to the ribosome Gamerdinger et al. 2015). The presence of a exit site, and many of these factors use partially not-yet-exposed signal anchor sequence inside overlapping binding surfaces on ribosomes. An the ribosomal tunnel increases the affinity of emerging question is how these factors gain reg- SRP for ribosomes, and the early binding of ulated accessto nascent polypeptidesand wheth- SRP to those RNCs depends on NAC (Berndt er they compete and/or collaborate at the ribo- et al. 2009; Zhang et al. 2012). This early SRP some exit site (Gamerdinger and Deuerling recruitment is likely mediated by subtle struc- 2014; Gloge et al. 2014). tural alterations of the ribosome and NAC at the The only tunnel exit-binding factors that are exit site such that SRP binding is favored. More- at least as abundant as ribosomes are TF in bac- over, in the absence of NAC, SRP partially binds teria and NAC in eukaryotes (Merz et al. 2006; to nonsecretory RNCs thereby perturbing the Raue et al. 2007). These major cotranslational accurate substrate selection of MAP1 by block- systems are supposed to play an important role ing its ribosomal access (del Alamo et al. 2011; in orchestrating nascent chain-processing events Nyathi and Pool 2015). Thus, although NAC at the ribosome exit site. A molecular interplay and MAP1 can bind simultaneously to ribo- between TF and SRP is well documented in vitro. somes and do not directly modulate each other’s TF generally prevents SRP binding to ribosomes, binding properties (Nyathi and Pool 2015), except to those RNCs presenting signal se- uncontrolled binding of a third factor can cause quences (Bornemann et al. 2014). This RNC unforeseen problems in protein biogenesis. This prefilter function by TF could explain why a example shows the delicateness of the molecular small amount of SRP (ratio of SRP to ribosome interplay at the tunnel exit and highlights the about 1:100) is sufficient for effective targeting of dynamic binding and release of ribosome-asso- membrane proteins (Jensen and Pedersen 1994). ciated protein biogenesis factors through a con- Moreover, TF also enhances the specificity of certed action. However, we are only beginning to SRP-dependent protein targeting. Both factors understand the complex interdependency of can bind simultaneously to signal sequence- these factors and how this affects the specific containing RNCs, and TF regulates SRP function and timely targeting of nascent chains into the at multiple targeting steps, including initial correct protein biogenesis pathway.

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Chaperone Interactions at the Ribosome

CONCLUDING REMARKS folded protein response, resulting in increased chaperone expression and apoptosis. PLoS ONE 7: e44038. doi:10 The myriad of recently accumulated data on the .1371/journal.pone.0044038 in vivo nascent substrates, structures, and mech- Balchin D, Hayer-Hartl M, Hartl FU. 2016. In vivo aspects of 353: anisms of ribosome-associated chaperones from protein folding and quality control. Science aac4354. doi:10.1126/science.aac4354 prokaryotic and eukaryotic kingdoms suggests Becker AH, Oh E, Weissman JS, Kramer G, Bukau B. 2013. that they are versatile and vital elements of the Selective ribosome profiling as a tool for studying the chaperone network crucial to control the quality interaction of chaperones and targeting factors with na- 8: and transport of newly synthesized proteins. In scent polypeptide chains and ribosomes. Nat Protoc 2212–2239. doi:10.1038/nprot.2013.133 addition, not discussed here at all, there is cul- Berndt U, Oellerer S, Zhang Y, Johnson AE, Rospert S. 2009. minating evidence that these chaperones may A signal-anchor sequence stimulates signal recognition display additional functions off the ribosome, particle binding to ribosomes from inside the exit tunnel. – Proc Natl Acad Sci 106: 1398–1403. doi:10.1073/pnas for example, TF and RAC Ssb have been sug- .0808584106 gested to promote the assembly of ribosomal Bloss TA, Witze ES, Rothman JH. 2003. Suppression of particles and NAC was found to enhance the CED-3-independent apoptosis by mitochondrial βNAC refolding of aggregated luciferase in C. elegans in Caenorhabditis elegans. Nature 424: 1066–1071. doi: (Martinez-Hackert and Hendrickson 2009; 10.1038/nature01920 Borgese N, Mok W, Kreibich G, Sabatini DD. 1974. Koplin et al. 2010; Kirstein-Miles et al. 2013). Ribosomal–membrane interaction: In vitro binding of ri- The future challenge will be to dissect their indi- bosomes to microsomal membranes. J Mol Biol 88: 559– vidual roles during protein biogenesis and gain a 580. doi:10.1016/0022-2836(74)90408-2 deeper structural and mechanistic understanding Bornemann T, Holtkamp W, Wintermeyer W. 2014. Inter- play between trigger factor and other protein biogenesis of their ribosomal and nonribosomal activities. factors on the ribosome. Nat Commun 5: 4180. doi:10 .1038/ncomms5180 Boyer LA, Latek RR, Peterson CL. 2004. The SANT domain: ACKNOWLEDGMENTS A unique histone-tail-binding module? Nat Rev Mol Cell Biol 5: 158–163. doi:10.1038/nrm1314 We apologize that we could not discuss all as- Buskiewicz I, Deuerling E, Gu SQ, Jockel J, Rodnina MV, pects of de novo folding and chaperone func- Bukau B, Wintermeyer W. 2004. Trigger factor binds to tions in depth. Moreover, we apologize to all our ribosome-signal-recognition particle (SRP) complexes colleagues whose research was not or only very and is excluded by binding of the SRP receptor. Proc Natl Acad Sci 101: 7902–7906. doi:10.1073/pnas.0402231101 briefly discussed or not cited. We thank Dr. fi Chen DH, Huang Y, Liu C, Ruan Y, Shen WH. 2014. Func- Christina Schlatter for assistance in gure prep- tional conservation and divergence of J-domain-contain- aration and Sandra Fries for proofreading the ing ZUO1/ZRF orthologs throughout evolution. Planta 239: – manuscript. M.G., S.G.K., and E.D. are support- 1159 1173. doi:10.1007/s00425-014-2058-6 fl ed by the Deutsche Forschungsgemeinschaft Conz C, Otto H, Peisker K, Gautschi M, Wol eT,Mayer MP, Rospert S. 2007. Functional characterization of (DFG)-funded collaborative research center the atypical Hsp70 subunit of yeast ribosome-associated SFB969 on the “Chemical and Biological Prin- complex. 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Chaperone Interactions at the Ribosome

Elke Deuerling, Martin Gamerdinger and Stefan G. Kreft

Cold Spring Harb Perspect Biol published online March 4, 2019

Subject Collection Protein Homeostasis

Proteome-Scale Mapping of Perturbed The Amyloid Phenomenon and Its Significance in Proteostasis in Living Cells Biology and Medicine Isabel Lam, Erinc Hallacli and Vikram Khurana Christopher M. Dobson, Tuomas P.J. Knowles and Michele Vendruscolo Pharmacologic Approaches for Adapting A Chemical Biology Approach to the Chaperome Proteostasis in the Secretory Pathway to in Cancer−−HSP90 and Beyond Ameliorate Protein Conformational Diseases Tony Taldone, Tai Wang, Anna Rodina, et al. Jeffery W. Kelly Cell-Nonautonomous Regulation of Proteostasis Proteostasis in Viral Infection: Unfolding the in Aging and Disease Complex Virus−Chaperone Interplay Richard I. Morimoto Ranen Aviner and Judith Frydman The Autophagy Lysosomal Pathway and The Proteasome and Its Network: Engineering for Neurodegeneration Adaptability Steven Finkbeiner Daniel Finley and Miguel A. Prado Functional Modules of the Proteostasis Network Functional Amyloids Gopal G. Jayaraj, Mark S. Hipp and F. Ulrich Hartl Daniel Otzen and Roland Riek Protein Solubility Predictions Using the CamSol Chaperone Interactions at the Ribosome Method in the Study of Protein Homeostasis Elke Deuerling, Martin Gamerdinger and Stefan G. Pietro Sormanni and Michele Vendruscolo Kreft Recognition and Degradation of Mislocalized Mechanisms of Small Heat Shock Proteins Proteins in Health and Disease Maria K. Janowska, Hannah E.R. Baughman, Ramanujan S. Hegde and Eszter Zavodszky Christopher N. Woods, et al. The Nuclear and DNA-Associated Molecular Structure, Function, and Regulation of the Hsp90 Chaperone Network Machinery Zlata Gvozdenov, Janhavi Kolhe and Brian C. Maximilian M. Biebl and Johannes Buchner Freeman

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