Ribosome-associated Complex Binds to Ribosomes in Close Proximity of Rpl31 at the Exit of the Polypeptide Tunnel in Yeast

Abstract

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. ⌬rpl31a⌬rpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, ⌬rpl31a⌬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 ⌬rpl31a⌬rpl31b⌬zuo1 did not exceed that of ⌬rpl31a⌬rpl31b or ⌬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 ⌬rpl31a⌬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. INTRODUCTIONTwo Hsp70 family members Ssb1/2 (Ssb1 and Ssb2 differ by only four amino acids) and Ssz1 and one J-domain protein (Zuo1) are abundant components of the translation machinery of Saccharomyces cerevisiae (Raue et al., 2007). The three chaperones are genetically linked and form a functional triad. Lack of either SSB1/2, SSZ1, or ZUO1 results in slow growth, cold sensitivity, and pronounced hypersensitivity against aminoglycosides such as paromomycin (Gautschi et al., 2002;Hundley et al., 2002). Ssz1 and Zuo1 assemble into a stable heterodimeric complex termed RAC (ribosome-associated complex). RAC acts as a cochaperone for Ssb1/2 and stimulates its ATP hydrolysis. The function requires both RAC subunits (Huang et al., 2005;Conz et al., 2007).RAC is anchored to the ribosome via Zuo1 (Gautschi et al., 2001). The idea is that positioning of RAC on the ribosome is required for its interaction with Ssb1/2 (Yan et al., 1998). However, the function of Ssz1 does not strictly depend on stable interaction with Zuo1 or ribosomes (Conz et al., 2007). How exactly Zuo1 anchors RAC is currently unclear. It was proposed that Zuo1 binds to ribosomes, in part, by interaction with rRNA (Yan et al., 1998). However, purified Zuo1 unspecifically interacts with a variety of nucleic acids. Initially, Zuo1 was identified via its ability to interact with Z-DNA (Zhang et al., 1992), it also interacts tightly with tRNA (Wilhelm et al., 1994) and recently was shown to bind to a small inhibitor RNA (Raychaudhuri et al., 2006). The mouse homolog MIDA1 interacts with DNA that forms small stem loop structures (Inoue et al., 2000). The diversity of nucleic acids that interact with Zuo1 raises the qu...

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

“…4). The level of small ribosomal subunits in the WT and ⌬Rpl31A/B strain are rather similar resulting in an accumulation of 40 S subunits, as also observed by Peisker et al (39). More importantly, in the WT strain roughly 50% of the cellular NAC cosediments with the ribosomes through the sucrose cushion at low salt conditions (100 mM KOAc), whereas in the ⌬Rpl31A/B strain only a very minor fraction of cellular NAC is detected in the pellet fraction.…”

“…However, RACs ribosome binding is most likely qualitatively different and may also include participation of the ribosomal RNA (39).…”

“…Strain MH272-3f␣ and the ⌬Rpl31A/B strain were provided by S. Rospert (39). Cloning was performed in E. coli XL1 Blue (Stratagene), expression in E. coli ER2566 (New England Biolabs).…”

“…To test this hypothesis we prepared S100 extracts from a ⌬Rpl31A/B strain and its maternal WT strain MH272-3f␣ (39). Both extracts were adjusted according to their general protein content judged by their absorption at 280 nm.…”

Ribosome Deficiency Protects Against ER Stress in Saccharomyces cerevisiae

“…Assigning exclusive functions to particular RPs, however, is complicated due to the highly cooperative nature of the interactions between RPs and the rRNA in the ribosome. For example, Rpl26, Rpl31, and Rpl39 all localize to the polypeptide tunnel exit of the ribosome (Ban et al 2000;Peisker et al 2008), and are each individually dispensable for viability. However, strains lacking both Rpl31 and Rpl39 are inviable (Peisker et al 2008), suggesting that these proteins function somewhat redundantly.…”