The nuclear‐coded chloroplast 22‐kDa heat‐shock protein of Chlamydomonas

Abstract

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlumydomonus reinhurdtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine a-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.In response to a sudden elevation of temperature, a number of specific genes, termed heat-shock genes, are activated in both procaryotic and eucaryotic cells [ 11. Following induction, a rapid transcription and preferential translation of the corresponding heat-shock mRNAs takes place and results in a significant accumulation of the heat-shock proteins [2]. In accordance with the universality of the heat-shock response, a relatively high degree of similarity characterizes heat-shock proteins from various organisms. In higher plants smallmolecular-mass heat-shock proteins (1 5 -30 kDa) represent a particularly prominent and heterogeneous group of proteins coded by multigene families [3 -61. The complexity and the diversity in the sizes of the low-molecular-mass heat-shock proteins seem to indicate a lower degree of similarity, in contrast to the high degree of conservation in the amino acid sequences of the high-molecular-mass heat-shock proteins common to both the plant and animal kingdoms. Like their high-molecular-mass counterparts, the small heat-shock proteins in plants are nuclear-coded and translated on cytosolic ribosomes. The majority of them remain in the cytosol and become associated with the cytoskeleton...

The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins

“…Like many other heat shock proteins, members of the alpha-crystallin family can be found in a variety of eukaryotes, i.e., in plant cells (18,32,36), various animal and insect cells (humans, hsp27; Drosophila sp., hsp26; spiny dogfish, alpha-crystallin) (11,22,24), and in yeast cells (hsp26) (5). However, in contrast to the 70K (DnaK and hsp70) and 60K (GroEL and hsp60) families, members of the alpha-crystallin family have not been reported as major proteins in prokaryotes (50).…”

“…A common feature of the low-molecular-weight heat shock proteins is a well-conserved short sequence near the C terminus with a derived consensus sequence, D/N-G-V-L-T-IIV-X-V/A (18), corresponding to a sequence in the alphacrystallin protein. Both the 14K protein of M. tuberculosis and the 18K protein of M. leprae contain this region, i.e., KGILTVSV, aa 118-125, and EGVLKLSI, aa 108-115, respectively (Fig.…”

“…Membrane translocation without processing is not unique to the 14K protein but has also been described for another alpha-crystallin heat shock protein, the chloroplast 22-kDa protein of Chlamydomonas sp. (18).…”

The Identification of a Heat-Shock Protein Complex in Chloroplasts of Barley Leaves

“…In C reinhardtii, preferential association of this protein with the thylakoid membranes was suggested to be with the grana stacks (8,12,34). This was corroborated by immunocytochemical localization of most of the 22-kD HSP in the granal fraction of thylakoid membranes isolated from heated cells (8).…”

“…2). Confirmation of the transcriptional origin of the HSP22 homolog of pea, soybean, maize, and C reinhardtii was obtained by the isolation of specific cDNA clones for each protein (8,23,40). However, only one study so far has reported chloroplast-encoded HSPs in higher plants (14), although no genes for such HSPs have yet been isolated.…”