Figure 4.

Coexpression of Hsp17 class CI prevents the formation of sedimentable HsfA2 complexes under control but not under heat stress conditions. Immunoblot analysis of HsfA2 expression and distribution between soluble (S₁₀₀) and insoluble (P₁₀₀) protein fractions after coexpression with the indicated forms of Hsp17-CI and/or Hsp17-CII in control (A) and heat stressed (B) protoplasts. To minimize possible interferences from the endogenous cellular heat stress response of the tobacco protoplasts, the inactive, 3HA-tagged version of HsfA2 was used. In some cases the plasmid mixture was complemented by addition of pRTLpHsfA1ΔC394 (samples 5–8 and 10 in part A, B). For heat stress treatment (B) protoplasts were incubated for 2 h at 40°C before harvesting. The analyzed proteins are indicated on the left margin by arrows and the corresponding antibodies. The open arrow head at the lower section in part B indicates the heat stress-induced expression of endogenous, tobacco Hsp17 class CI proteins. Variations in the steady state levels of HsfA2 expression were estimated on the basis of immunoblot signals in the WPE fractions and the values given at the bottom of the corresponding immunoblots are normalized to the expression levels in samples number 2 (=1.0) in A and B, respectively. For evaluation of the amounts of insoluble HsfA2 in the P₁₀₀ fractions see legend to Figure 3.