RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin

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.

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 (Δzuo), IDA2 (Δssz1), IDA12 (Δzuo1Δssz1), 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.