Abstract
In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.
Highlights
Molecular life at the interface of the cell membrane and the cytoplasmic milieu may be regulated by proteins that attach to and detach from the membrane surface in response to signals
The major protein we obtained from Paramecium by this approach was not an annexin but a novel protein with two copies of the C2 domain and one copy of a domain related to the A domain that mediates protein-protein interactions between integrins and their extracellular ligands
Isolation of a 55-kDa Phospholipid-binding Protein from Extracts of Paramecium—Calcium-dependent, phospholipid-binding proteins were isolated from the soluble fraction of homogenates of mass cultures of P. tetraurelia by binding to multilamellar vesicles prepared from brain lipid extracts enriched in phosphatidylserine
Summary
Molecular life at the interface of the cell membrane and the cytoplasmic milieu may be regulated by proteins that attach to and detach from the membrane surface in response to signals. Two major protein motifs that regulate calciumdependent interactions with membrane lipids have been extensively characterized: The annexin fold [1, 2], and the C2 domain [3, 4]. The absence of clear enzymatic activities has made the functions of the annexins difficult to determine Their interaction with membranes can lead to modulation of other membranebinding proteins such as phospholipases [11]. They exhibit a “bivalent” activity in the sense that they can bind to two membranes and draw them together into a complex that is subject to fusion with additional perturbation of membrane structure [2, 12, 13]. Because the Paramecium protein associates with lipid membranes, like a “companion,” we have given the protein a name reflecting this property: copine (pronounced “ko-peenЈ”), from the French feminine noun copine, which means “friend.”
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