Protein refolding in peroxisomes is dependent upon an HSF1-regulated function

Lonneke Heldens,Jurre Hageman,Siebe T Van Genesen,Lars L.P Hanssen,Nicolette H Lubsen,Harm H Kampinga

doi:10.1007/s12192-012-0335-5

Abstract

Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of HSF1-regulated chaperones, blocked post-heat shock refolding of luciferase targeted to the cytoplasm, nucleus, or peroxisomes, while refolding of endoplasmic reticulum (ER)-targeted luciferase was inhibited by about 50 %. Luciferase refolding in the cytoplasm could be partially restored by expression of HSPA1A and fully by both HSPA1A and DNAJB1. For full refolding of ER luciferase, HSPA1A expression sufficed. Neither nuclear nor peroxisomal refolding was rescued by HSPA1A. A stimulatory effect of DNAJB1 on post-heat shock peroxisomal luciferase refolding was seen in control cells, while refolding in the cytoplasm or nucleus in control cells was inhibited by DNAJB1 expression in the absence of added HSPA1A. HSPB1 also improved refolding of peroxisomal luciferase in control cells, but not in dnHSF1 expressing cells. HSP90, HSPA5, HSPA6, and phosphomevalonate kinase (of which the synthesis is also downregulated by dnHSF1) had no effect on peroxisomal refolding in either control or chaperone-depleted cells. The chaperone requirement for post-heat shock refolding of peroxisomal luciferase in control cells is thus unusual in that it can be augmented by DNAJB1 or HSPB1 but not by HSPA1A; in dnHSF1 expressing cells, expression of none of the (co)-chaperones tested was effective, and an as yet to be identified, HSF1-regulated function is required.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-012-0335-5) contains supplementary material, which is available to authorized users.

Highlights

Proteins are made as linear chains but are active as intricately folded units often associated in larger assemblies
We found that expression of dominant negative heat shock factor 1 (HSF1) mutant (dnHSF1) needed to be induced for at least 48 h to see a decrease in the post-heat shock refolding of cytoplasmic luciferase
The fact that there is a delayed response to induction of dnHSF1 indicates that it is not the expression of dnHSF1 itself that leads to a reduced refolding capacity but that it is a secondary effect of the expression of dnHSF1, such as chaperone depletion

Summary

Introduction

Proteins are made as linear chains but are active as intricately folded units often associated in larger assemblies. The three main cellular compartments, cytoplasm, mitochondria, and ER, each have a compartment dedicated general chaperoning network. These networks are similar in that they all contain related chaperones and associated factors which aid in folding (Hsp and Hsp machines; Welch and Feramisco 1982; Mayer and Bukau 2005), chaperones which deliver substrates to the folding machines (DNAJ/Hsp proteins), and chaperones which can store unfolded protein for later refolding or degradation (the small heat shock proteins) (Vos et al 2008). In spite of the lack of classical chaperones, heat-denatured peroxisomal luciferase is refolded as efficiently as it is in other cellular compartments (Hageman et al 2007), suggesting that peroxisomes can deal with unfolded proteins

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Conclusion