RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin - PubMed (original) (raw)
RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin
M Gautschi et al. Proc Natl Acad Sci U S A. 2001.
Abstract
The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family. Our current understanding of which homologs functionally interact is incomplete. Zuotin is a DnaJ homolog bound to the yeast ribosome. We have now identified the DnaK homolog Ssz1p/Pdr13p as zuotin's partner chaperone. Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP. In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperone-like effect on nascent chains. In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo. However, overexpression of zuotin partly rescued the growth defect of the Delta ssz1 strain, whereas overexpression of Ssz1p did not affect the Delta zuo1 strain, suggesting a pivotal function for the DnaJ homolog.
Figures
Figure 1
Purification of RAC. (A) Yeast RAC was purified as described in Materials and Methods. The purification was monitored by SDS/PAGE and staining with Coomassie blue. Lanes represent proteins released from ribosomes by treatment with 700 mM Kacetate (salt wash); the pool of the active fractions after separation of the salt wash on a ResourceQ (resourceQ); the pool of the active fractions after separation on Superdex200 (superdex200); and the pool of the active fractions after separation on MonoQ (monoQ). Molecular mass standards are indicated on the left. (B) Purified RAC is free of Ssb1/2p. Aliquots of the samples shown in_A_ were analyzed by immunoblotting with antibodies specific for Ssz1p, zuotin, and Ssb1/2p.
Figure 2
RAC stimulates translocation of RNCs into mitochondria. Ribosome-bound Mdh1p was generated in the presence of [35S]methionine in a yeast translation extract derived from the ΔNAC strain YRG16. RNCs were isolated under high-salt conditions, with factors stimulating translocation removed, and subsequently incubated with isolated mitochondria. After the reaction mitochondria were reisolated, equal aliquots of mitochondrial pellets, containing mitochondria-bound RNCs, were resuspended, precipitated with trichloroacetic acid, and analyzed by SDS/PAGE, followed by fluorography. STD 10%, 10% of the RNCs added per assay. p, Mdh1p precursor; m, mature Mdh1p. (A) Purified RAC stimulates translocation in a dose-dependent manner. High-salt-washed ΔNAC-RNCs were preincubated in the absence (−) or in the presence of purified RAC. The concentration of RAC is given as the final concentration in the assay. Translocation efficiency is given as the percentage of the total amount of RNCs added to the assay (Lower). (B) RAC and NAC synergistically stimulate translocation. The translocation assay was performed as in A, except that high-salt-washed RNCs were preincubated in the absence (−) or in the presence of purified RAC (RAC), a combination of RAC and NAC (RAC + NAC), or NAC (NAC). The final concentrations in the translocation assay were 100 nM for RAC and 40 nM for NAC, respectively.
Figure 3
RAC is a stable heterodimer. (A) RAC is stable in the presence of adenine nucleotides. Proteins released from ribosomes by 700 mM Kacetate were used for immunoprecipitations in the presence of 2 mM ATP (ATP) or 2 mM ADP (ADP) or in the absence of nucleotide (−) with antisera directed against zuotin (α zuotin) or Ssz1p (α Ssz1p), or with preimmune serum (PI). After incubation for 3 h at 4°C, bound proteins were recovered in the pellet (P) and unbound proteins in the supernatant (S), and both fractions were analyzed by SDS/PAGE and immunoblotting with antibodies specific for Ssa1/2p, Ssz1p, zuotin, or Ssb1/2p as indicated. The total of the ribosomal salt wash added to each single reaction is shown on the left (T). (B) Molecular mass determination of RAC was performed by sedimentation equilibrium measurements at 10,000 rpm and 20°C in an analytical ultracentrifuge. The experimental data could be fitted to a homogeneous population of particles with an apparent mass of 126 kDa. (Upper) Experimental data and fit (−). (Lower) Deviation of fit and experimental data.
Figure 4
Binding of RAC to ribosomes. (A) The major fraction of RAC is bound to ribosomes in a salt-sensitive manner. Translation-competent cytosol (C) was separated into a postribosomal supernatant (S) and a ribosomal pellet (P) in the presence of either 120 mM Kacetate (low salt) or 700 mM Kacetate (high salt). (B) RAC is reversibly released by high concentrations of salt. After release of endogenous RAC from the ribosome (high-salt P), the ribosomal pellet was resuspended under low-salt conditions, and purified RAC was added to a final concentration of 150 nM (high-salt P + RAC). After incubation for 5 min on ice the ribosomes (P2) were separated from the supernatant (S2) for a second time. As a control, RAC was treated the same, but in the absence of ribosomes (RAC). (C) Binding of RAC to ribosomes requires zuotin. Cytosol of MH272–3f (wild type), IDA12–2μ-SSZ1, and IDA12–2μ-ZUO1 were separated into postribosomal supernatant and ribosomal pellet in the presence of 120 mM Kacetate (low salt) or 700 mM Kacetate (high salt). Note that both Ssz1p and zuotin partly degrade during extract preparation in the absence of their partner protein. (A–C) Corresponding amounts of cytosol, supernatant, and ribosomal pellet were separated by SDS/PAGE and analyzed by immunodecoration, with the use of antibodies specifically recognizing Ssz1p, zuotin, Rpl16a, hexokinase (hexo), Ssa1/2p, Ssb1/2p, and αNAC.
Figure 5
Deletions in SSZ1 and ZUO1 display a similar but nonidentical phenotype. Yeast strains IDA1 (Δ_zuo_), IDA2 (Δ_ssz1_), IDA12 (Δ_zuo1_Δ_ssz1_), wild type (MH272–3fα), wt-2μ-SSZ1 (MH272–3fα overexpressing_SSZ1_), wt-2μ-ZUO1 (MH272–3fα overexpressing_ZUO1_), IDA12–2μ-SSZ1 (IDA12 overexpressing_SSZ1_), and IDA12–2μ-ZUO1 (IDA12 overexpressing_ZUO1_) were grown to early log phase at 30°C on minimal glucose medium. (A) Equal amounts of total yeast protein were separated on SDS/PAGE followed by immunodecoration with antibodies directed against Ssz1p, zuotin, and, as a control for loading of equal amounts, malate dehydrogenase (mMDH). (B) Serial 5-fold dilutions of early log-phase cultures. Dilutions containing the same number of cells were spotted from top to bottom onto rich glucose-containing medium (YPD, yeast extract/peptone/dextrose). The time and temperature of incubation are given. RT, room temperature; d, days; paro, 75 μg/ml paromomycin.
Similar articles
- A functional chaperone triad on the yeast ribosome.
Gautschi M, Mun A, Ross S, Rospert S. Gautschi M, et al. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4209-14. doi: 10.1073/pnas.062048599. Proc Natl Acad Sci U S A. 2002. PMID: 11929994 Free PMC article. - Zuotin, a ribosome-associated DnaJ molecular chaperone.
Yan W, Schilke B, Pfund C, Walter W, Kim S, Craig EA. Yan W, et al. EMBO J. 1998 Aug 17;17(16):4809-17. doi: 10.1093/emboj/17.16.4809. EMBO J. 1998. PMID: 9707440 Free PMC article. - Structural analysis of the ribosome-associated complex (RAC) reveals an unusual Hsp70/Hsp40 interaction.
Fiaux J, Horst J, Scior A, Preissler S, Koplin A, Bukau B, Deuerling E. Fiaux J, et al. J Biol Chem. 2010 Jan 29;285(5):3227-34. doi: 10.1074/jbc.M109.075804. Epub 2009 Nov 17. J Biol Chem. 2010. PMID: 19920147 Free PMC article. - Two chaperones locked in an embrace: structure and function of the ribosome-associated complex RAC.
Zhang Y, Sinning I, Rospert S. Zhang Y, et al. Nat Struct Mol Biol. 2017 Aug 3;24(8):611-619. doi: 10.1038/nsmb.3435. Nat Struct Mol Biol. 2017. PMID: 28771464 Review. - DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70.
Cyr DM, Langer T, Douglas MG. Cyr DM, et al. Trends Biochem Sci. 1994 Apr;19(4):176-81. doi: 10.1016/0968-0004(94)90281-x. Trends Biochem Sci. 1994. PMID: 8016869 Review.
Cited by
- Oxidative protein folding in the intermembrane space of human mitochondria.
Zarges C, Riemer J. Zarges C, et al. FEBS Open Bio. 2024 Oct;14(10):1610-1626. doi: 10.1002/2211-5463.13839. Epub 2024 Jun 12. FEBS Open Bio. 2024. PMID: 38867508 Free PMC article. Review. - Chaperone Hsp70 helps Salmonella survive infection-relevant stress by reducing protein synthesis.
Chan C, Groisman EA. Chan C, et al. PLoS Biol. 2024 Apr 4;22(4):e3002560. doi: 10.1371/journal.pbio.3002560. eCollection 2024 Apr. PLoS Biol. 2024. PMID: 38574172 Free PMC article. - NAC and Zuotin/Hsp70 chaperone systems coexist at the ribosome tunnel exit in vivo.
Ziegelhoffer T, Verma AK, Delewski W, Schilke BA, Hill PM, Pitek M, Marszalek J, Craig EA. Ziegelhoffer T, et al. Nucleic Acids Res. 2024 Apr 12;52(6):3346-3357. doi: 10.1093/nar/gkae005. Nucleic Acids Res. 2024. PMID: 38224454 Free PMC article. - Exploration of the truncated cytosolic Hsp70 in plants - unveiling the diverse T1 lineage and the conserved T2 lineage.
Chen YJ, Cheng SY, Liu CH, Tsai WC, Wu HH, Huang MD. Chen YJ, et al. Front Plant Sci. 2023 Nov 16;14:1279540. doi: 10.3389/fpls.2023.1279540. eCollection 2023. Front Plant Sci. 2023. PMID: 38034583 Free PMC article. - Prions in Microbes: The Least in the Most.
Son M, Han S, Lee S. Son M, et al. J Microbiol. 2023 Oct;61(10):881-889. doi: 10.1007/s12275-023-00070-4. Epub 2023 Sep 5. J Microbiol. 2023. PMID: 37668956 Review.
References
- Agashe V R, Hartl F U. Semin Cell Dev Biol. 2000;11:15–25. - PubMed
- Bukau B, Deuerling E, Pfund C, Craig E A. Cell. 2000;101:119–122. - PubMed
- Schatz G, Dobberstein B. Science. 1996;271:1519–1526. - PubMed
- Rassow J, Voos W, Pfanner N. Trends Cell Biol. 1995;5:207–212. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Miscellaneous