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

• Published in: The Plant journal : for cell and molecular biology Vol. 24; no. 3; pp. 397 - 411
• Main Authors: KIRSCHNER, Marc, WINKELHAUS, Sybille, THIERFELDER, Jörg M, NOVER, Lutz
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell Science 01.11.2000
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Cell physiology; Cells, cell elements: structure and function; Dimerization; Drosophila - genetics; Fundamental and applied biological sciences. Psychology; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - genetics; Heat-Shock Proteins - metabolism; Hot Temperature; Interactions. Associations; Intermolecular phenomena; Macromolecular Substances; Mice; Microscopy, Electron; Molecular biophysics; Molecular Sequence Data; Nicotiana - genetics; Nicotiana - metabolism; Oligodeoxyribonucleotides - genetics; Pisum sativum - genetics; Plant physiology and development; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified; Plants, Toxic; Protein Structure, Quaternary; Protoplasts - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Transformation, Genetic; Transient response; Oligomerization; Gene expression; Nicotiana tabacum; Stress; Aggregation; Molecular assembly; Dicotyledones; Protoplast; Angiospermae; Heat shock protein; Spermatophyta; Transgenic plant; Property structure relationship; Solanaceae; Experimental plant; Protein body
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313x.2000.00887.x

Heat-stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat-stressed 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-specific 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 auto-aggregation and for recruitment of class I proteins. The class-specific 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.