Heat Shock Protein 90 Mediates Protein-protein Interactions between Human Aminoacyl-tRNA Synthetases

Jeongwoo Kang,Taeho Kim,Young-Gyu Ko,Seung Bae Rho,Sang Gyu Park,Min Jung Kim,Ho Jeong Kwon,Sunghoon Kim

doi:10.1074/jbc.m909965199

Jeongwoo Kang, Taeho Kim + Show 6 more

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https://doi.org/10.1074/jbc.m909965199

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Journal: Journal of Biological Chemistry	Publication Date: Oct 1, 2000
Citations: 35	License type: cc-by

Affiliation: Sejong University

Abstract

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

Highlights

Mammalian aminoacyl-tRNA synthetases (ARSs)1 are unique in their formation of a macromolecular complex
The same cells expressing LexA-isoleucyl-tRNA synthetase (IRS)-C and B42 were treated with these drugs and cultivated in the yeast medium with leucine. The growth of these cells was only slightly affected by the treatment with these drugs (Fig. 4, middle). These results suggest that the interaction of EPRS and IRS is affected by the inhibition of hsp90, further supporting that this interaction is mediated by hsp90
Hsp90 is one of the most abundant cellular proteins, it is highly restrictive in target selection under normal physiological conditions

Summary

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Assay—To identify proteins interacting with the three repeats of EPRS, the cDNA encoding the peptide from Val573 to Lys889 of EPRS (EPRS-L) was subcloned into pLex202 vector using EcoRI and SalI sites and used as bait to screen a human HeLa cDNA library expressing B42 fusion proteins [19]. The in vitro translated EPRS-L was mixed with the Escherichia coli protein extract containing GST-hsp or GST in binding buffer composed of 40 mM HEPES, pH 7.6, 20% glycerol, 1 mM DTT, 0.3 ␮M phenylmethylsulfonyl fluoride, and protease inhibitor mixture (pepstatin A, leupeptin, antipain, and chymostatin; 5 ␮g/ml each). To determine the induction of the leu gene, the EGY48 strain expressing LexA-IRS-C (from Glu966 to Phe1266) and B42-EPRS-L was cultivated in 5 ml of yeast synthetic medium (UraϪ, HisϪ, TrpϪ, 2% glucose) at 30 °C overnight with shaking. The growth of the EGY48 strain alone or expressing LexA-IRS-C and B42 in the presence of these antibiotics was determined using the same methods, except that the cells were cultivated in yeast synthetic medium containing leucine. Autoradiography was quantified using a phosphor image analyzer (BAS-3000; Fuji)

RESULTS

Findings

DISCUSSION