Interaction between the N-terminal and Middle Regions Is Essential for the in Vivo Function of HSP90 Molecular Chaperone

Akio Mizuno,Kumiko Oi,Etsuko Tanaka,Ichiro Yahara,Takashi Takagi,Takao Ayuse,Shigeki Matsumoto,Yoko Kimura,Jun Imai,Takayuki K. Nemoto,Toshio Ono,Takeshi Kobayakawa

doi:10.1074/jbc.m203038200

Abstract

At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400). The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.

Highlights

We recently proposed that the liberation of the N-terminal client-binding region from the middle suppressor region is the mechanism underlying the temperature-dependent activation of HtpG, an E. coli homologue of mammalian HSP90 [30]
To make the interpretation simple, we here used the regions of HSP90a instead of the full-length form
Limited proteolysis of Region A with trypsin produced a limited number of proteolytic fragments (Fig. 1a)

Summary

Introduction

The 90-kDa heat shock protein (HSP90) has been demonstrated to be an important molecule, chaperoning a variety of cellular proteins, such as steroid receptors [1,2,3], protein kinases involved in signal transduction [4,5,6], and even retrovirus reverse transcriptase [7] and endothelial nitric oxide synthase (8, and see reviews 9, 10).HSP90 occupies a central part of the chaperone network, the “foldsome,” and functions in cooperation with other chaperones and co-chaperones, such as immunophilins, CDC37/p50, HSP70, p23, Hip, Hop/p60 and PA28 (11-13 and see reviews 9, 10).This assembly process of the HSP90-substrate protein complex requires ATP [14, 15], which induces a conformational change in HSP90 [16,17,18].Recently, it was demonstrated that HSP90 is capable of linking substrates for degradation by the ubiquitin-proteasome pathway, by co-operating with the E3 ligase CHIP [19,20,21]. HSP90 may play a central role in deciding the fate of proteins, refolding or degradation.HSP90-family proteins are composed of 3 regions at the primary structure level [22, 23]. HSP90 occupies a central part of the chaperone network, the “foldsome,” and functions in cooperation with other chaperones and co-chaperones, such as immunophilins, CDC37/p50, HSP70, p23, Hip, Hop/p60 and PA28 (11-13 and see reviews 9, 10). This assembly process of the HSP90-substrate protein complex requires ATP [14, 15], which induces a conformational change in HSP90 [16,17,18]. It was demonstrated that HSP90 is capable of linking substrates for degradation by the ubiquitin-proteasome pathway, by co-operating with the E3 ligase CHIP [19,20,21].

Results

Discussion

Conclusion