Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast.

The guided entry of tail-anchored proteins (GET) pathway assists in the posttranslational delivery of tail-anchored proteins, containing a single C-terminal transmembrane domain, to the ER. Here we uncover how the yeast GET pathway component Get4/5 facilitates capture of tail-anchored proteins by Sgt2, which interacts with tail-anchors and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of tail-anchored proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting. Exposure of internal transmembrane domains at the tunnel exit induces high-affinity ribosome binding of SRP, which in turn prevents ribosome binding of Get4/5. In this way, the position of a transmembrane domain within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit.

The ribosome-associated complex RAC serves in a relay that directs nascent chains to Ssb.

The conserved ribosome-associated complex (RAC) consisting of Zuo1 (Hsp40) and Ssz1 (non-canonical Hsp70) acts together with the ribosome-bound Hsp70 chaperone Ssb in de novo protein folding at the ribosomal tunnel exit. Current models suggest that the function of Ssz1 is confined to the support of Zuo1, however, it is not known whether RAC by itself serves as a chaperone for nascent chains. Here we show that, via its rudimentary substrate binding domain (SBD), Ssz1 directly binds to emerging nascent chains prior to Ssb. Structural and biochemical analyses identify a conserved LP-motif at the Zuo1 N-terminus forming a polyproline-II helix, which binds to the Ssz1-SBD as a pseudo-substrate. The LP-motif competes with nascent chain binding to the Ssz1-SBD and modulates nascent chain transfer. The combined data indicate that Ssz1 is an active chaperone optimized for transient, low-affinity substrate binding, which ensures the flux of nascent chains through RAC/Ssb.

A dual role of the ribosome-bound chaperones RAC/Ssb in maintaining the fidelity of translation termination.

The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.

Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain.

Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.

Cotranslational Intersection between the SRP and GET Targeting Pathways to the Endoplasmic Reticulum of Saccharomyces cerevisiae.

Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K(+) channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation.

Release factor eRF3 mediates premature translation termination on polylysine-stalled ribosomes in Saccharomyces cerevisiae.

Ribosome stalling is an important incident enabling the cellular quality control machinery to detect aberrant mRNA. Saccharomyces cerevisiae Hbs1-Dom34 and Ski7 are homologs of the canonical release factor eRF3-eRF1, which recognize stalled ribosomes, promote ribosome release, and induce the decay of aberrant mRNA. Polyadenylated nonstop mRNA encodes aberrant proteins containing C-terminal polylysine segments which cause ribosome stalling due to electrostatic interaction with the ribosomal exit tunnel. Here we describe a novel mechanism, termed premature translation termination, which releases C-terminally truncated translation products from ribosomes stalled on polylysine segments. Premature termination during polylysine synthesis was abolished when ribosome stalling was prevented due to the absence of the ribosomal protein Asc1. In contrast, premature termination was enhanced, when the general rate of translation elongation was lowered. The unconventional termination event was independent of Hbs1-Dom34 and Ski7, but it was dependent on eRF3. Moreover, premature termination during polylysine synthesis was strongly increased in the absence of the ribosome-bound chaperones ribosome-associated complex (RAC) and Ssb (Ssb1 and Ssb2). On the basis of the data, we suggest a model in which eRF3-eRF1 can catalyze the release of nascent polypeptides even though the ribosomal A-site contains a sense codon when the rate of translation is abnormally low.

Interaction of nascent chains with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae.

As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39 expose domains to the interior of the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunnel proteins and nascent chains. As model proteins we employed Dap2, which contains a hydrophobic signal anchor (SA) segment, and the chimera Dap2α, in which the SA was replaced with a hydrophilic segment, with the propensity to form an α-helix. Employing a newly developed FLAG exposure assay, we find that the nascent SA segment but not the hydrophilic segment adopted a stable, α-helical structure within the tunnel when the most C-terminal SA residue was separated by 14 residues from the peptidyl transferase center. Using UV cross-linking, antibodies specifically recognizing Rpl17 or Rpl39, and a His6-tagged version of Rpl4, we established that all three tunnel proteins of yeast contact the SA, whereas only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the tunnel exposed domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with nascent chain residues throughout the ribosomal tunnel.

NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum.

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

The chaperone network connected to human ribosome-associated complex.

Mammalian ribosome-associated complex (mRAC), consisting of the J-domain protein MPP11 and the atypical Hsp70 homolog (70-homolog) Hsp70L1, can partly complement the function of RAC, which is the homologous complex from Saccharomyces cerevisiae. RAC is the J-domain partner exclusively of the 70-homolog Ssb, which directly and independently of RAC binds to the ribosome. We here show that growth defects due to mRAC depletion in HeLa cells resemble those of yeast strains lacking RAC. Functional conservation, however, did not extend to the 70-homolog partner of mRAC. None of the major human 70-homologs was able to complement the growth defects of yeast strains lacking Ssb or was bound to ribosomes in an Ssb-like manner. Instead, our data suggest that mRAC was a specific partner of human Hsp70 but not of its close homolog Hsc70. On a mechanistic level, ATP binding, but not ATP hydrolysis, by Hsp70L1 affected mRAC's function as a J-domain partner of Hsp70. The combined data indicate that, while functionally conserved, yeast and mammalian cells have evolved distinct solutions to ensure that Hsp70-type chaperones can efficiently assist the biogenesis of newly synthesized polypeptide chains.

Ribosome-associated complex binds to ribosomes in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast.

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Delta rpl31a Delta rpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Delta rpl31a Delta rpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Delta rpl31a Delta rpl31b Delta zuo1 did not exceed that of Delta rpl31a Delta rpl31b or Delta zuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Delta rpl31a Delta rpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.

Functional characterization of the atypical Hsp70 subunit of yeast ribosome-associated complex.

Eukaryotic ribosomes carry a stable chaperone complex termed ribosome-associated complex consisting of the J-domain protein Zuo1 and the Hsp70 Ssz1. Zuo1 and Ssz1 together with the Hsp70 homolog Ssb1/2 form a functional triad involved in translation and early polypeptide folding processes. Strains lacking one of these components display slow growth, cold sensitivity, and defects in translational fidelity. Ssz1 diverges from canonical Hsp70s insofar that neither the ability to hydrolyze ATP nor binding to peptide substrates is essential in vivo. The exact role within the chaperone triad and whether or not Ssz1 can hydrolyze ATP has remained unclear. We now find that Ssz1 is not an ATPase in vitro, and even its ability to bind ATP is dispensable in vivo. Furthermore, Ssz1 function was independent of ribosome-associated complex formation, indicating that Ssz1 is not merely a structural scaffold for Zuo1. Finally, Ssz1 function in vivo was inactivated when both nucleotide binding and Zuo1 interaction via the C-terminal domain were disrupted in the same mutant. The two domains of this protein thus cooperate in a way that allows for severe interference in either but not in both of them.

The chaperones MPP11 and Hsp70L1 form the mammalian ribosome-associated complex.

Soluble Hsp70 homologs cotranslationally interact with nascent polypeptides in all kingdoms of life. In addition, fungi possess a specialized Hsp70 system attached to ribosomes, which in Saccharomyces cerevisiae consists of the Hsp70 homologs Ssb1/2p, Ssz1p, and the Hsp40 homolog zuotin. Ssz1p and zuotin are assembled into a unique heterodimeric complex termed ribosome-associated complex. So far, no such specialized chaperones have been identified on ribosomes of higher eukaryotes. However, a family of proteins characterized by an N-terminal zuotin-homology domain fused to a C-terminal two-repeat Myb domain is present in animals and plants. Members of this family, like human MPP11 and mouse MIDA1, have been implicated in the regulation of cell growth. Specific targets of MPP11/MIDA1, however, have remained elusive. Here, we report that MPP11 is localized to the cytosol and associates with ribosomes. Purification of MPP11 revealed that it forms a stable complex with Hsp70L1, a distantly related homolog of Ssz1p. Complementation experiments indicate that mammalian ribosome-associated complex is functional in yeast. We conclude that despite a low degree of homology on the amino acid level cooperation of ribosome-associated chaperones with the translational apparatus is well conserved in eukaryotic cells.