Abstract
Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.
Highlights
Calcium ions, present inside all eukaryotic cells, are important second messengers in the transduction of biological signals
We found that the putative calmodulin-binding domain of Ssb1/Hsp75 shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse Hsp70 [25], which does not conform to any of the three classes of calmodulin-binding motifs described previously [4] and has been classified as a divergent calmodulin-binding motif restricted to this protein
From mammalian cells, where the primary Ca2+ stores are the endoplasmic or sarcoplasmic reticulum, the calcium influx in S. cerevisiae comes from the vacuole and a protein homologue of transient receptor potential channels - known as Yvc1p - is required for this release [29]
Summary
Present inside all eukaryotic cells, are important second messengers in the transduction of biological signals. The information encoded in the transient increase in Ca2+ concentration is transduced by intracellular Ca2+-binding proteins, which convert the Ca2+ signal to a wide variety of biochemical changes. Upon sequestering Ca2+, calmodulin changes its conformation and reveals two methionine-rich hydrophobic patches that interact with calmodulin-binding domains in a variety of proteins [2], regulating their activities. A program using algorithms based on these motifs is available in the Calmodulin Target Database (calcium.oci.utoronto.ca/) but it works by analyzing one sequence at a time, which prevents a global analysis of an entire genome. Sequence searches based on these criteria are not sufficient to identify unequivocally calmodulin target proteins and different analytical techniques, including calmodulin affinity chromatography and the gel overlay assay using labeled calmodulin, should be used in order to identify new activities and processes which are targets for Ca2+/calmodulin and to confirm potential candidates identified by proteomic tools
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