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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Ca(2+)-dependent ubiquitination of calmodulin in yeast. 130 6

In plants Ca2+ plays a crucial role as second messenger. Thus calmodulin is one of the most important signal transducing molecules for metabolic regulation in plants. Previously we showed that bovine testis calmodulin can be covalently coupled at one site to ubiquitin in a Ca2(+)-dependent manner in the presence of ATP/Mg2+ by ubiquityl-calmodulin synthetase. Since calmodulin from spinach has 13 amino acid sequence differences to bovine calmodulin - two of them in Ca2(+)-binding loops - it was unclear, whether a conjugation of ubiquitin to this molecule would be possible. In this paper it is shown that calmodulin from spinach and a similar calmodulin from the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2(+)-dependent manner. It is shown that higher molecular mass conjugates containing up to three ubiquitin molecules per calmodulin are obtained. Experiments with methylated ubiquitin demonstrate that, as with vertebrate calmodulins, only one lysine residue is linked to ubiquitin and that the incorporation of additional ubiquitin molecules leads to a polyubiquitin chain.
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PMID:Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2(+)-dependent manner. 217 31

Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.
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PMID:The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitin-binding first component, a ubiquitin-activating enzyme. 971 91

Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.
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PMID:The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties. 971 92