Functional Rescue of Mutant Human Cystathionine β-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae

Laishram R Singh,Warren D Kruger

doi:10.1074/jbc.m806387200

Abstract

Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine beta-synthase protein, I278T, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to I278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of I278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper I278T folding. In contrast, deletion of HSP26 results in increased I278T protein and activity, whereas overexpression of Hsp26 results in reduced I278T protein. The Hsp26-I278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded.

Highlights

We found that addition of up to 6% ethanol to standard yeast media allowed growth of I278T expressing yeast in SCϪCys media in a concentration-dependent manner (Fig. 1A)
We focused on SSA2, because it was the only one of the three genes thought to be present in the cytosol. (The SSQ1 and SSC1 are thought to encode mitochondrial proteins [22].) to determine whether cytosolic Hsp70 is playing a crucial role in proper I278T folding, we deleted SSA2 in a strain expressing I278T and measured CBS function by examining growth on cysteine-free media in the presence and absence of 4% ethanol (Fig. 2B)
The data presented here show that substantial enzymatic function could be restored to human I278T CBS expressed in S. cerevisiae by either reduction of Hsp26 or increase in Hsp70 levels

Summary

Introduction

A, saturated culture of yeast strain WY35 expressing I278T CBS (WY35 pI278T) was diluted 1:1000 in SCϪCys media with the indicated amount of ethanol at 30 °C for 24 h. Addition of 4% ethanol caused a 280% increase in steady-state levels of I278T protein, as well as a 930% increase in CBS enzyme activity (Fig. 1B). Examination of CBS mRNA levels by quantitative real time PCR showed that ethanol had no effect on mRNA levels (data not shown), indicating that the increase in steady-state I278T levels was because of stabilization of the protein.

Results

Conclusion