Ca(2+)-dependent ubiquitination of calmodulin in yeast.
Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.
Jennissen
H P
HP
Institut für Physiologische Chemie, Universität-GHS-Essen, Germany.
Botzet
G
G
Majetschak
M
M
Laub
M
M
Ziegenhagen
R
R
Demiroglou
A
A
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D010453:Peptide Synthases / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D002147:Calmodulin; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; C059404:ubiquitin-calmodulin ligase; D010764:Phosphorylase Kinase; D044764:Ubiquitin-Activating Enzymes; D010453:Peptide Synthases; D002118:Calcium