HSP90 homologue from Madagascar periwinkle (Catharanthus roseus): cDNA sequence, regulation of protein expression and location in the endoplasmic reticulum.
We describe cDNAs for a HSP90 homologue from Catharanthus roseus and studies on the regulation of expression. The largest cDNA (2670 bp) coded for a protein of 817 amino acids with a calculated size of 93,491 Da and a pI of 4.61. It contained a eucaryotic secretory signal, the endoplasmic reticulum (ER) targeting and retention signal (Lys-Asp-Glu-Leu), and the HSP90 protein family signature with one conservative exchange (Asn-Lys-Asp-Ile-Phe-Leu instead of Asn-Lys-Glu-Ile-Phe-Leu). RNA blots revealed a transcript of 2.8-2.9 kb, and genomic DNA blots suggested a single gene. The expression was analysed with antiserum against a fusion protein expressed in Escherichia coli. Immunoblots revealed a protein of 93 +/- 1.5 kDa (often a doublet) only in the membrane fraction, and sucrose density gradients suggested association with the ER. The protein was constitutively expressed in C. roseus cell cultures grown at 25 degrees C, and expression was apparently unaffected by various stress conditions, such as heat, high sucrose, elicitor from Phytophthora megasperma or yeast extract. It was not detectable in young C. roseus plants at room temperature, and heat shock for several hours at 37 degrees C was necessary to obtain detectable expression. In maize (Zea mays), a cross-reacting protein was detectable in cell cultures, but not in young plants. The results suggested that the cloned protein is not a major component in the heat shock response. We propose a chaperone role in the assembly and processing of cell wall components and other secreted proteins, i.e. functions that are very active in cells with a high rate of growth and division.
Schröder
G
G
Institut für Biologie II, Universität Freiburg, Germany.
Beck
M
M
Eichel
J
J
Vetter
H P
HP
Schröder
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D018833:Chaperonins; D003001:Cloning, Molecular; D004247:DNA; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010946:Plants, Medicinal / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D006360:Heat-Shock Proteins; D010940:Plant Proteins; D011506:Proteins; D004247:DNA; D018833:Chaperonins