The Hsp60-(p.V98I) Mutation Associated with Hereditary Spastic Paraplegia SPG13 Compromises Chaperonin Function Both in Vitro and in Vivo

Peter Bross,Søren Naundrup,Jakob Hansen,Marit Nyholm Nielsen,Jane Hvarregaard Christensen,Mogens Kruhøffer,Johan Palmfeldt,Thomas Juhl Corydon,Niels Gregersen,Debbie Ang,Costa Georgopoulos,Kåre Lehmann Nielsen

doi:10.1074/jbc.m800548200

Abstract

We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harbors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.

Highlights

While analyzing a French family with autosomal dominant pure hereditary spastic paraplegia SPG13, we detected heterozygosity for a mutation (c.292G Ͼ C; p.V98I) in the HSPD1 gene, which encodes the mitochondrial Hsp60 chaperonin [6]
Based on our findings the V98I mutation affects the ATPase activity and results in a dramatically decreased folding activity of complexes consisting of the mutant subunits only
In contrast to an artificially constructed ATPase mutant, the V98I mutation exerts no salient dominant negative effect when co-expressed with wildtype Hsp60

Summary

Introduction

While analyzing a French family with autosomal dominant pure hereditary spastic paraplegia SPG13, we detected heterozygosity for a mutation (c.292G Ͼ C; p.V98I) in the HSPD1 gene, which encodes the mitochondrial Hsp60 chaperonin [6]. Strain was subsequently transformed with a second plasmid, a derivative of pBAD/hisA (Invitrogen), that contains an operon expressing Hsp10 and either wild-type or different mutant variants of Hsp60 under control of the arabinose-inducible BAD promoter (Fig. 2A). These cells carry a deletion of the essential endogenous groES/groEL genes and are maintained alive by a plasmid expressing an IPTG-inducible operon encoding human Hsp10 and the mature form of wild-type Hsp60 (Fig. 2A, upper panel).

Results

Conclusion