SummaryHeat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heatstressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat-stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG-like complexes. Class I and class II small Hsps formed class-speci®c dodecamers of 210±280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG-like complexes (auto-aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C-terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced autoaggregation and for recruitment of class I proteins. The class-speci®c formation of dimers as a prerequisite for oligomerization was analysed by the yeast two-hybrid system. In the presence of the endogenous (tobacco) set of heat-stress-induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo-oligomers of 200±700 kDa, none of them could be recruited to HSG complexes.

A 68-kDa heat-stress protein (HSP68) has been purified from cell-suspension cultures of tomato (Lycopersicon peruvianum L.). Antibodies raised against HSP68 cross-react with the Escherichia coli heat-stress protein DnaK. HSP68 was found to be a hydrophilic, ATP-binding protein. Immunological analysis of subcellular fractions and immunogold-labelling of ultrathin sections showed consistently that HSP68 is localized in the mitochondrial matrix. In-vitro translation experiments indicated that HSP68 is synthesized as a precursor protein. Immunoscreening of cDNA libraries from tomato and potato (Solanum tuberosum L.) led to the isolation of corresponding cDNA clones. The deduced amino-acid sequences show strong relationships to the DnaK-like proteins from bacteria and organelles of eukaryotic cells. The protein HSP68 is constitutively expressed, but its synthesis is increased during heat stress in all cells of higher plants investigated so far.

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