Ansgar Koplin1, Marc Erhardt1,2 and Elke Deuerling1*
1Laboratory of Molecular Microbiology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
2 present address: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, Utah 84112, USA.
* Corresponding author: email: Elke.Deuerling@uni-konstanz.de, phone:+49-7531-882647, fax: :+49-7531-884036
Running title: Dual function of ribosome-associated chaperones
Keywords: Ribosomes, nascent polypeptides, co-translational protein folding, Hsp70, protein homeostasis
Total number of characters: 52.881
During eukaryotic protein synthesis a ribosome-tethered Hsp70/40-chaperone system composed of RAC (ribosome-associated complex) and Ssb in yeast contacts emerging nascent polypeptides to support early folding steps. We demonstrate that Ssb/RAC cooperates with the nascent polypeptide-associated complex (NAC) in co-translational protein folding.
Simultaneous deletions of genes encoding NAC and Ssb caused a severe synthetic sickness of cells grown at 30°C and the loss of cell viability under protein folding stress conditions.
Deprivation of Ssb impaired NAC association with translating ribosomes and provoked aggregation of newly synthesized proteins, which was enhanced by additional deletion of NAC. Moreover, nac∆ssb∆ cells revealed the formation of ribosomal half-mers and a pronounced deficiency of 60S particles accompanied by strongly reduced amounts of translating ribosomes and of total cellular protein indicating that both chaperone systems are additionally involved in ribosome biogenesis. Our findings are consistent with a model where a dual function of ribosome-associated chaperones physically connects ribosome biogenesis and chaperone-assisted protein folding providing a new concept to balance protein folding with global protein synthesis and to adjust protein homeostasis.
In all cells, the folding of newly synthesized proteins requires the assistance of a chaperone network. At the forefront are ribosome-associated chaperones, which contact the nascent polypeptide to control co-translational protein folding and prevent unwarranted aggregation or degradation of newly synthesized proteins. Subsequently, additional cytosolic chaperones including members of the Hsp70- and Hsp60-chaperone families bind to a subset of newly made proteins to further support de novo folding processes (Bukau et al., 2000; Frydman, 2001; Hartl and Hayer-Hartl, 2002; Wegrzyn and Deuerling, 2005). While in bacteria a single ribosome-associated chaperone, Trigger Factor, is known to assist co-translational protein folding, other ribosome-associated systems have evolved in eukaryotes that are not related to Trigger Factor and functionally less well understood. Eukaryotic ribosomes are in association with a Hsp70/40-based chaperone machine and with the nascent polypeptide-associated complex NAC (Fig. 1A). Both systems are conserved and abundant components of the eukaryotic cytosol, which dynamically bind to the large ribosomal subunit and interact with nascent polypeptides early during biogenesis (Hundley et al., 2005; Pfund et al., 1998; Raue et al., 2007; Rospert et al., 2002; Wegrzyn and Deuerling, 2005).
The Hsp70/40-system is tripartite in yeast (Fig. 1A) and consists of the Hsp70-chaperone Ssb and the complex RAC (ribosome-associated complex), a stable heterodimer formed by the Hsp70-homolog Ssz and the Hsp40 Zuotin. Ssb binds to the 60S ribosomal subunit and is the only component of the Hsp70/40 system that contacts nascent polypeptides via its substrate-binding domain (Pfund et al., 1998). Two Ssb proteins (Ssb1 and Ssb2) exist in yeast which are functionally interchangeable and differ by only four amino acids (Ssb hereafter). The efficient interaction of Ssb with nascent chains depends on the presence of RAC (Gautschi et al., 2002; Hundley et al., 2002; Nelson et al., 1992; Pfund et al., 1998). RAC binds to ribosomes via Zuotin and both subunits are required to stimulate the ATP hydrolysis of Ssb thereby promoting substrate binding (Gautschi et al., 2001; Huang et al., 2005; Hundley et al., 2005; Otto et al., 2005). Yeast strains lacking either one or all three components of the chaperone triad show similar pleiotropic phenotypes, like hypersensitivity to a wide range of cations, including cationic aminoglycosides that inhibit translation, and sensitivity to high salt concentrations and to low temperatures (Gautschi et al., 2001; Hundley et al., 2002; Kim and Craig, 2005; Yan et al., 1998).
2002). While both subunits were shown to crosslink to nascent polypeptides (Wiedmann et al., 1994), only the β-NAC subunit contacts the ribosome at the ribosomal protein L23 (corresponds to Rpl25 in yeast) via a predicted loop region which includes the conserved consensus motif RRK (X)n KK, located between two predicted alpha-helices in its N-terminus (Fig. 1A). A triple mutation in this conserved NAC-ribosome binding motif (RRK to AAA) abolished ribosome binding both in vitro and in vivo (Wegrzyn et al., 2006). Furthermore, this NAC-RRK/AAA variant could no longer crosslink to nascent polypeptides implying that the ribosomal attachment of NAC is crucial for its interaction with nascent polypeptides.
Multiple potential functions for NAC have been proposed so far. NAC has been suggested to stimulate the import of fumarase in vivo (Yogev et al., 2007) and of the malate dehydrogenase precursor into yeast mitochondria in vitro (Fünfschilling and Rospert, 1999). Furthermore, a role for NAC in controlling ribosome-targeting and protein translocation to the endoplasmic reticulum (ER) has been proposed. However, these functions of NAC are a matter of debate since no consistent data could be obtained (Raden and Gilmore, 1998) (Lauring et al., 1995) (Powers and Walter, 1996) (Moller et al., 1998) (Wiedmann and Prehn, 1999). Given its ability to associate with ribosomes and nascent polypeptides, additionally a chaperone-like function of NAC has been suggested albeit no data supporting this assumption exist that far and up to date the in vivo function of NAC is barely understood (Bukau et al., 2000; Frydman, 2001; Hartl and Hayer-Hartl, 2002; Wegrzyn and Deuerling, 2005). In Saccharomyces cerevisiae, the EGD2 gene encodes the α-NAC subunit of NAC and two genes, EGD1 and BTT1, encode β-NAC subunits with BTT1 being expressed at very low levels compared to EGD1. Yeast cells in which NAC-encoding genes were deleted altogether are viable and lack any noticeable phenotype (Reimann et al., 1999).
We set out to investigate the in vivo functions of the ribosome-associated systems Ssb/RAC and NAC in yeast. In particular, we addressed the question whether NAC is involved in co-translational protein folding and functionally connected with Ssb/RAC. To this end we combined knockout mutations in genes encoding NAC and Ssb/RAC and characterized the phenotypes and consequences on protein folding. We found that Ssb/RAC and NAC genetically interact with each other and functionally cooperate during de novo folding. This result strongly supports a chaperone-like function of NAC in the intricate chaperone network
Synergistic growth defects of cells simultaneously lacking NAC and Ssb
To investigate whether the two ribosome-associated systems NAC and the Ssb/RAC (Fig. 1A) are interconnected and cooperate in the chaperone network of the eukaryotic cytosol, we combined knock-out mutations of all three genes encoding NAC subunits (egd1∆, btt1∆ and egd2∆ referred to hereafter as nac∆) with deletions of the two Ssb genes (ssb1∆, ssb2∆
referred to hereafter as ssb∆). Cells carrying the quintuple knockout mutations (nac∆ssb∆
cells) were viable, however, revealed a severe synthetic sickness. At 30°C nac∆ssb∆ cells grew significantly slower than nac∆ or ssb∆ cells as judged by the colony size on plates (Fig.
1B) and the extended doubling time in liquid cultures (Fig. 1C). Moreover, the application of low concentrations of drugs that impair protein synthesis or folding such as the arginine analog L-canavanine or the translation inhibitor Hygromycin B, resulted in a severe drop of cell viability of nac∆ssb∆ cells and the plating efficiency decreased by several orders of magnitude as compared to control cells of wt, nac∆ or ssb∆ (Fig. 1B). As reported earlier, cells lacking only Ssb revealed slower growth at 30°C in the presence or absence of drugs compared to wild type (wt) cells, while cells lacking NAC showed no growth impairment (Fig. 1B,C) (Kim and Craig, 2005; Reimann et al., 1999).
Importantly, expression of wt NAC from a centromeric plasmid fully complemented the phenotype of nac∆ssb∆ cells back to the character of a ssb∆ strain, while the expression of the NAC-RRK/AAA ribosome-binding mutant did not (Fig. 1B). This implies that the observed phenotype is specific for NAC and critically depends on its ribosome association. The synthetic sickness and synergistic phenotype of cells lacking simultaneously NAC and Ssb was reproducibly observed in another yeast background (Fig. 1D) excluding clone or strain specific defects and suggesting general synthetic defects in the absence of these ribosome-associated proteins. Moreover, to confirm that the phenotype of nac∆ssb∆ cells is related to Ssb chaperone function in the triad, nac∆ mutations were combined with a zuo∆ mutation.
This knock-out combination resulted in similar synthetic defects as compared with nac∆ssb∆
cells (suppl. Fig. 1A). We also observed that the cold sensitivity as well as the salt sensitivity is more pronounced in cells lacking Ssb and NAC compared to cells lacking only Ssb (data not shown).
In summary, the synergistic growth defects of nac∆ssb∆ cells reveal a genetic interaction between NAC and Ssb suggesting that the two ribosome-associated systems in eukaryotes work in parallel or in partly overlapping pathways during de novo protein folding. The data
Loss of NAC and Ssb affects the folding of newly synthesized polypeptides but does not provoke a heat shock response
To investigate whether the folding of newly synthesized polypeptides is affected by the loss of the two ribosome-associated systems, cells lacking either one or both systems were grown at 30°C in defined media without methionine until log phase and then pulsed for 1 min with
35S-methionine to label newly synthesized proteins. Subsequently, translation was stopped by the addition of cycloheximide and aggregated proteins in these cells were isolated from lysates by sedimentation analysis. Although we noticed that lysates prepared from ssb∆ and ssb∆nac∆ cells always contained significantly lower yields of total protein, 35S-Met had been incorporated equally well into translated polypeptides as judged by the similar labeling efficiency visualized in 15 µg of total lysate by autoradiography (Fig. 2A, lanes 1-4). Neither wt cells nor cells lacking NAC revealed a substantial amount of insoluble material, which is consistent with the lack of any nac∆ phenotype (Fig. 2A, lanes 5+6). In contrast, the absence of Ssb caused aggregation of a variety of newly made polypeptides. This aggregation tendency was more pronounced in cells lacking both Ssb and NAC (Fig. 2 A, lanes 7+8). The aggregates included nascent polypeptides as well as proteins translated and released within the labeling period explaining the mixture of a radioactive smear with discrete bands.
Quantification of the aggregated 35S-containing material showed that about 2.5% of newly synthesized proteins were aggregation-prone in the absence of NAC and Ssb, while only about 1.3% of proteins were insoluble in the absence of Ssb (Fig. 2A, lower panel). Thus, there is a synergistic defect of cells lacking NAC and Ssb in the folding of newly made proteins. However, this defect is surprisingly small and affects only a minor fraction of de novo synthesized proteins, suggesting that compensatory mechanisms may exist in nac∆ssb∆
cells which prevent cells from massively accumulating misfolded proteins.
One plausible explanation is that cells lacking ribosome-associated chaperones may induce a heat shock response in order to increase the chaperone capacity and the proteolytic activity of the cell, thereby minimizing protein aggregation. To examine this possibility, we determined the amount of cytosolic Hsp70-proteins Ssa1-4, including the heat shock inducible paralogs Ssa3 and Ssa4, and of heat inducible Hsp104 in wt and mutant cells grown under permissive temperature at 30°C or exposed to heat shock treatment. As evident from Fig. 2 B, the levels of Ssa and Hsp104 proteins were similar at 30°C in all strains tested. In contrast, a
response (Fig. 2B). Thus, the loss of Ssb and NAC did not provoke the induction of a heat shock response at permissive temperature. The low amount of aggregated proteins detected in nac∆ssb∆ cells at 30°C is therefore not explicable by a compensatory heat shock response.
Ribosomal particles aggregate in cells lacking Ssb and NAC
To better understand the cellular consequences caused by the loss of Ssb and NAC, we investigated whether insoluble material accumulated in these cells to an extent that allowed isolation and identification by SDS-PAGE and mass spectrometry. To this end we prepared aggregates from logarithmically growing wt and mutant cells in quantitative amounts and separated the isolated insoluble fractions by SDS-PAGE for Coomassie-staining (Fig. 3A). In agreement with results above no pronounced protein aggregation was found in wt or nac∆
cells, while the insoluble fractions prepared from cells lacking Ssb revealed a pattern of aggregated proteins mainly consisting of small-sized species with a molecular weight between 17 and 55 kDa (Fig. 3A). Interestingly, the pattern of aggregated proteins detected by Coomassie-staining was distinct from the insoluble protein repertoire detected upon 35S-Met pulse labeling (compare Fig. 2A with 3A). This difference can be explained by the fact that the 35S-pulse-labeled aggregated material exclusively visualized newly synthesized proteins, while the Coomassie-stained aggregates represented insoluble material found under steady state conditions also including pre-existing, insoluble proteins. In line with the data presented above, aggregation was more pronounced in cells lacking Ssb and NAC, while the pattern of aggregated protein species remained similar in ssb∆ and nac∆ssb∆ cells, suggesting that the loss of NAC affects a similar set of proteins (Fig. 3A). The major bands visible in the insoluble fraction prepared from nac∆ssb∆ cells were excised from the Coomassie-stained gel and identified by mass spectrometry (Tab. 1). A total of 64 proteins could be unambiguously identified including predominantly ribosomal proteins (52 out of 64) from both ribosomal subunits. Moreover, the insoluble material contained some ribosomal biogenesis factors such as Tif6, Nog1 and Rlp24, the elongation factor EF-1a (Tef2), and chaperones including the Hsp40-homologs Ydj1 and Sis1, a subunit (Cct8) of the prefoldin chaperone and small heat shock protein Hsp26 (Tab. 1). To confirm the aggregation of ribosomal proteins, we performed Western blot analysis of the isolated insoluble fractions with antibodies directed against Rpl35, a ribosomal protein of the large subunit identified by mass spectrometry in the insoluble fraction. Concordantly with the analysis above, we detected the aggregation of
Three different and not mutually exclusive scenarios could explain the aggregation of mainly ribosomal proteins in these mutant cells: (i) Ribosomal proteins might be particularly aggregation-prone in cells lacking ribosome-attached chaperones and thus aggregate upon synthesis prior to their engagement in ribosome assembly, or (ii) nascent chains aggregate during translation and thereby pull ribosomes into the insoluble fraction. Since nascent chains are heterogenous, only the ribosomal proteins are visible as distinct bands in the Coomassie-stained gel. (iii) Finally, Ssb and NAC may also play a role in the biogenesis of ribosomes besides their function in chaperoning nascent polypeptides. Loss of these chaperones may render pre-ribosomal particles and their associated biogenesis factors susceptible for aggregation.
First, we investigated whether actively translating ribosomes are trapped in the aggregates via misfolded nascent polypeptides and included a treatment of the lysate with puromycin to cause the release of nascent chains from ribosomes prior to isolation of aggregates. However, the puromycin treatment turned out to be very inefficient in lysates prepared from cells lacking Ssb and NAC for unknown reasons and thus no reliable data could be derived.
Nevertheless, we consider it as very likely that some translating ribosomes are trapped in the aggregates given the finding that also elongation factor EF-1α (Tef2) is found in the pellet fraction of nac∆ssb∆ cells. Moreover, some chaperones are present in the insoluble material, including a subunit (Cct8) of prefoldin, which is known to bind to nascent polypeptides (Hansen et al., 1999). It is likely that these chaperones are trapped into aggregates by binding to their substrates as described for other protein aggregation in chaperone deficient cells (Deuerling et al., 2003; Houry et al., 1999) and thus supporting the assumption that the aggregation of translating ribosomes caused by the misfolding of nascent polypeptides contributed to the aggregated material.
Secondly, we analyzed the aggregates for the presence of RNA and ribosomal particles.
Ethidium bromide (EtBr) staining of the isolated insoluble fractions in the SDS-PAGE revealed the presence of nucleic acids as a smear in all fractions that contained aggregated ribosomal proteins (Fig. 2B). A treatment of the aggregated fractions with RNaseA prior to EtBr treatment completely abolished staining, while a treatment with DNaseI did not. Thus, we assume that the intense smear of nucleic acid detected in these fractions most likely represents ribosomal RNA, which had been fragmented during our isolation procedure (Fig.
dispersed aggregates (Fig. 3C). Compared to a negative-stained EM-image of total ribosomes (composed of 40S, 60S, 80S and polysomes) isolated from wt cells, some of the aggregated particles matched in their sizes with some ribosomal particles present in the ribosomal fraction (Fig. 3C, white arrows).
Based on these data, we conclude that the aggregates isolated from nac∆ssb∆ cells are likely composed of ribosomal particles rather than individual aggregation-prone proteins. In summary, these results support the idea that the absence of Ssb and NAC provokes the aggregation of ribosomal particles.
Loss of Ssb and NAC functions leads to the formation of ribosomal half-mers and decreases the amount of 60S ribosomal subunits and total cellular protein
Based on the observation that ribosomal particles and some ribosomal maturation factors are present in the aggregated fraction prepared from nac∆ssb∆ cells, we next examined whether the lack of Ssb and NAC caused a defect in ribosomal biogenesis. To this end, we compared the ribosomal profiles from wt cells with the profiles from cells lacking NAC and Ssb by separating the total lysate on a sucrose gradient using ultracentrifugation and a subsequent profile readout of the gradient at 254 nm. Importantly, the peak heights as detected by A254nm
were sensitive indicators of the levels of each ribosomal species, since the same amount of sample was loaded. As evident from the ribosomal profile (Fig. 4A), the absence of Ssb, and much more pronounced the simultaneous loss of Ssb and NAC, had severe consequences on the ribosomal species found in the cytosol of these cells: in ssb∆ cells, and more distinct in nac∆ssb∆ cells, the formation of ribosomal half-mers was visible by a shoulder in the 80S and polysome peaks (Fig. 4A, indicated by arrows). Ribosomal half-mers are frequently observed in cells that are impaired in the maturation of 60S ribosomal subunits. Immature 60S particles are known to be inactive in translation and thus do not per se disturb the translation process but produce ribosomal species with an uncomplexed 40S portion and a lower amount of actively translating ribosomes (Basu et al., 2001; Sydorskyy et al., 2003). Indeed, the peak ratio of 40S to 60S was inversed in ∆nac∆ssb cells compared to wt cells due to a severe decrease of the 60S species (Fig. 4B). Additionally, the 80S peak was significantly lower (Fig. 4C) and the number of polysomal peaks was reduced in the nac∆ssb∆ profile compared to the wt (Fig. 4A). Comparable to the effects of NAC and Ssb on the folding of newly synthesized proteins, the 60S subunit deficiency was already apparent in the absence of Ssb.
To confirm the finding that NAC and Ssb affect the amount of 60S ribosomal subunits, we quantified the total amount of 60S particles in wt and diverse mutant cells by analyzing the ribosomal fraction of total lysates derived from similar cell numbers with antibodies directed against two large ribosomal proteins Rpl25 (Fig. 5A) and Rpl35 (Suppl. Fig.1B). We found that the total amount of 60S particles was decreased by more than 50% in ssb∆nac∆ cells compared to wt. A deficiency in the amount of 60S particles was also visible in cells lacking only Ssb or any other triad component in the presence or absence of the NAC system, however, in part not as strong as for nac∆ ssb∆ knockout cells. The deficit of 60S particles
To confirm the finding that NAC and Ssb affect the amount of 60S ribosomal subunits, we quantified the total amount of 60S particles in wt and diverse mutant cells by analyzing the ribosomal fraction of total lysates derived from similar cell numbers with antibodies directed against two large ribosomal proteins Rpl25 (Fig. 5A) and Rpl35 (Suppl. Fig.1B). We found that the total amount of 60S particles was decreased by more than 50% in ssb∆nac∆ cells compared to wt. A deficiency in the amount of 60S particles was also visible in cells lacking only Ssb or any other triad component in the presence or absence of the NAC system, however, in part not as strong as for nac∆ ssb∆ knockout cells. The deficit of 60S particles