The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step.
A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii 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 alpha-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.
Grimm
B
B
Institut für Botanik, Universität Hannover, Federal Republic of Germany.
Ish-Shalom
D
D
Even
D
D
Glaczinski
H
H
Ottersbach
P
P
Ohad
I
I
Kloppstech
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D002696:Chlamydomonas / Q000235:genetics*; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D005815:Genetic Code; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011061:Poly A / Q000302:isolation & purification; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification*; D014158:Transcription, Genetic
D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA