Transient expression and heat-stress-induced co-aggregation of endogenous and heterologous small heat-stress proteins in tobacco protoplasts

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone comple xes found in all heat-stressed plant cells. We have developed an experiment al system involving expression of cytosolic class I and class II small heat -stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplas ts to study the structural prerequisites for the assembly of HSG or MSG-lik e complexes. Class I and class II small Hsps formed class-specific dodecame rs of 210-280 kDa, which, upon heat stress, were incorporated into HSG comp lexes. Interestingly, class II dodecamers alone could form MSG-like complex es (auto-aggregation), whereas class I dodecamers could do so only in the p resence of class II proteins (recruitment). By analysing C-terminal deletio n forms of Hsp17 class II, we obtained evidence that the intact C-terminus is critical for the oligomerization state, for the heat-stress-induced auto aggregation and for recruitment of class I proteins. The class-specific for mation 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 protei ns were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Alth ough other, more distantly related, members of the Hsp20 family, i.e. the p lastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well exp ressed in tobacco protoplasts and formed homo-oligomers of 260-700 kDa, non e of them could be recruited to HSG complexes.