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. 2001 Feb 19;152(4):851-6.
doi: 10.1083/jcb.152.4.851.

An essential role for the substrate-binding region of Hsp40s in Saccharomyces cerevisiae

J L Johnson  1 , E A Craig
Affiliations

An essential role for the substrate-binding region of Hsp40s in Saccharomyces cerevisiae

J L Johnson et al. J Cell Biol. .

Abstract

In addition to regulating the ATPase cycle of Hsp70, a second critical role of Hsp40s has been proposed based on in vitro studies: binding to denatured protein substrates, followed by their presentation to Hsp70 for folding. However, the biological importance of this model is challenged by the fact that deletion of the substrate-binding domain of either of the two major Hsp40s of the yeast cytosol, Ydj1 and Sis1, leads to no severe defects, as long as regions necessary for Hsp70 interaction are retained. As an in vivo test of this model, requirements for viability were examined in a strain having deletions of both Hsp40 genes. Despite limited sequence similarity, the substrate-binding domain of either Sis1 or Ydj1 allowed cell growth, indicating they share overlapping essential functions. Furthermore, the substrate-binding domain must function in cis with a functional Hsp70-interacting domain. We conclude that the ability of cytosolic Hsp40s to bind unfolded protein substrates is an essential function in vivo.

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Figures

Figure 1
Figure 1
The COOH terminus of either Sis1 or Ydj1 is essential. (A) Domains of Ydj1 and Sis1. Ydj1: J domain (J), G/F region (G/F), cysteine-rich region (cysteine-rich), COOH terminus (C-terminus), and farnesylation signal (F). Sis1: J domain (J), G/F region (G/F), G/M region (G/M), domains I (I) and II (II) of the COOH terminus, and dimerization domain (D). (B) Strain JJ1146 (sis1 ydj1/YCp50:SIS1) was transformed with indicated combinations of plasmids containing wild-type YDJ1 (or SIS1) and truncation mutants ydj1-134 (or sis1-121). After overnight incubation at 23°C, equal amounts of cells were plated onto selective media to maintain wild-type SIS1 (+SIS1) or media containing 5-FOA to counterselect for SIS1 (−SIS1). Plates were incubated at 23°C for 5 d.
Figure 3
Figure 3
Essential region of the COOH terminus of Ydj1. Strain JJ1146 (ydj1 sis1/YCp50:SIS1) expressing sis1-121 was transformed with wild-type YDJ1 or the indicated ydj1 mutants. (A) After overnight incubation at 23°C, equal amounts of cells were plated onto selective media to maintain wild-type SIS1 (+SIS1) or media containing 5-FOA to counterselect for SIS1 (−SIS1). Plates were grown at 23°C for 5 d. (B) Colonies that appeared on 5-FOA plate (sis1-121) were grown overnight and serial dilutions were grown at 30°C for 2 d on selective media (left). Growth of ydj1 mutants on selective media after 2 d at 30°C in a ydj1 disruption strain (WT SIS1) (right). WT, wild type.
Figure 2
Figure 2
Essential region of the COOH terminus of Sis1. Strain JJ1146 (ydj1 sis1/YCp50:SIS1) expressing ydj1-134 was transformed with wild-type SIS1 or sis1 mutants expressing Sis1 fragments of increasing length. (A) After overnight incubation at 23°C, equal amounts of cells were plated onto selective media to maintain wild-type SIS1 (+SIS1) or media containing 5-FOA to counterselect for SIS1 (−SIS1). Plates were grown at 23°C for 5 d. (B) Colonies that appeared on 5-FOA plates were grown overnight, and serial dilutions were grown at 30°C for 2 d on selective media (top). Yeast extracts expressing wild-type or mutant Sis1 proteins were separated on a 15% SDS–acrylamide gel, transferred to nitrocellulose, and immunoblotted with an antibody against Sis1 (bottom). (C) Structure of substrate-binding region of Sis1. Sites of truncation mutants are labeled and marked with arrows. Shaded residues are postulated to interact with bound substrate (Sha et al. 2000). Image was generated using MSI WebLab™ ViewerPro. C-term, COOH terminus; N-term, NH2 terminus; WT, wild type.
Figure 4
Figure 4
The COOH terminus must cooperate with a functional J domain in cis. Strain JJ1146 (ydj1 sis1/YCp50:SIS1) expressing sis1-121 and ydj1-134 from Ycplac22-134/121 was transformed with plasmids expressing the indicated sis1 or ydj1 mutant. Viability was assayed by plating cells in the presence of 5-FOA to counterselect for wild-type SIS1.
Figure 5
Figure 5
Partial rescue of ydj1 defects by YYS-262. (A) Hap1 was assayed in the ydj1 hem1 disruption strain JJ261 expressing full-length Ydj1, Ydj1-104, YYS-206, YYS-262, or overexpressing wild-type SIS1. β-Galactosidase activity of the UAS1/CYC1-lacZ reporter construct was measured in cells after overnight incubation at 25°C in the presence of high concentrations of ALA (250 μg/ml). (B) The ydj1 disruption strain JJ160 was transformed with plasmids expressing the indicated proteins. After overnight incubation at 25°C, 10-fold serial dilutions of cells were plated on rich media. Plates were grown for 2 d at the indicated temperatures.
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