Disease relevance of CMD1

• By fusing a portion of the Staphylococcus aureus spa gene (encoding protein A) to CMD1, the S. cerevisiae gene encoding calmodulin, we have generated a yeast calmodulin with an affinity tag able to bind immunoglobulins [1].
• Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11-yeast genomic DNA phage clones initially selected by the ability to bind 125I-labelled yeast CaM [2].
• Second, we have purified mammalian calmodulin from an overproducing Escherichia coli strain [3].
• Nucleotide sequence of the Bacillus anthracis edema factor gene (cya): a calmodulin-dependent adenylate cyclase [4].
• B. anthracis EF was not related to the Escherichia coli or yeast adenylate cyclases, but was related to the Bordetella pertussis calmodulin-dependent adenylate cyclase [4].

High impact information on CMD1

• Calcineurin is a eukaryotic Ca(2+)- and calmodulin-dependent serine/threonine protein phosphatase [5].
• The gene (designated CMD1) is a unique, single-copy locus, contains no introns, and resides on chromosome II [6].
• The amino acid sequence of yeast calmodulin shares 60% identity with other calmodulins [6].
• Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles [7].
• Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing [8].

Biological context of CMD1

• Although hCaM shares only 60% amino acid sequence identity with yeast calmodulin (CMD1 gene product), hCaM was able to substitute functionally for Cmd1 in yeast cells [9].
• The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1 [10].
• Molecular analysis of yeast chromosome II between CMD1 and LYS2: the excision repair gene RAD16 located in this region belongs to a novel group of double-finger proteins [11].
• Calmodulin mutants that have lost their high-affinity calcium binding sites are, however, able to carry out endocytosis normally [12].
• Calcium-independent calmodulin requirement for endocytosis in yeast [12].

Anatomical context of CMD1

• Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division [13].
• Both the actin and microtubule defects of arc35-1 can be suppressed by overexpression of calmodulin [14].
• Saccharomyces cerevisiae Arc35p works through two genetically separable calmodulin functions to regulate the actin and tubulin cytoskeletons [14].
• Calmodulin, a cytoplasmic calcium-binding protein, is indispensable for eukaryotic cell growth [15].
• Calmodulin binds tightly to vacuoles upon Ca2+ release [8].

Associations of CMD1 with chemical compounds

• Examination of 14 temperature-sensitive yeast mutants bearing one or more phenylalanine to alanine substitutions in the single essential calmodulin gene of yeast (CMD1) revealed diverse essential functions [15].
• The mutant carries two mutations in CMD1, isoleucine 100 is changed to asparagine and glutamic acid 104 is changed to valine [16].
• Third, Myo2p coimmunoprecipitates with calmodulin in the presence of Ca2+ or EGTA [17].
• The cysteine 911 to arginine mutation resides in the calmodulin-binding site and alone confers a temperature-sensitive phenotype [18].
• When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml [19].

Physical interactions of CMD1

• The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure [20].
• Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein [21].
• Co-immunoprecipitation, two-hybrid and overlay assays demonstrated that Cmd1p interacts with Myo5p, a type I unconventional myosin [22].
• Binding of centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components Kar1p and Spc110p [23].
• Calmodulin could bind to Myo1p through the IQ domain at the neck [24].

Enzymatic interactions of CMD1

• A putative ubiquitin protein ligase (E3-CaM) which cooperates with UBC4 in selectively ubiquitinating calmodulin has been partially purified from Saccharomyces cerevisiae [25].

Regulatory relationships of CMD1

• The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1 [26].
• Analysis of a collection of temperature-sensitive cmd1 mutants for their ability to suppress either the endocytic defect and/or the actin defect indicates that the two defects are tightly coupled [27].
• Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism [28].
• The cmdl-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter [29].
• An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca2 +-independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants [30].

Other interactions of CMD1

• A mutation in Myo2p, an unconventional myosin that is a possible target of calmodulin, did not inhibit endocytosis [12].
• Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1 [28].
• The solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p) [20].
• The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the K(d) for binding is 150 nM [20].
• To analyze other components involved in actin organization through calmodulin, we isolated and characterized mutations that show synthetic lethal interaction with cmd1-226; these "cax" mutants fell into five complementation groups [21].

Analytical, diagnostic and therapeutic context of CMD1

• Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggesting an importance of hydrophobic interaction between calmodulin and Myo2p [21].
• Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature [26].
• Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies [31].
• Immunoelectron microscopy presented here shows that calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque [18].
• Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells [32].

References