Abstract
BackgroundCopines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA – cpnF.ResultsIndependent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P.ConclusionsOur studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.
Highlights
Copines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes
Copines localize to the plasma membrane and intracellular membranes in methanol fixed cells To investigate the intracellular location of copines in Dictyostelium, the cDNAs for cpnA, cpnB, cpnC, cpnE, and cpnF were subcloned into the pTX-green fluorescent protein (GFP) vector at the SacI site for the overexpression of copines tagged with GFP at the N-terminus (GFP-Cpn) and cloned into the KpnI site for the overexpression of copines tagged with GFP at the C-terminus (Cpn-GFP)
We showed that CpnA with GFP at the N-terminus (GFP-CpnA) primarily localized to the cytoplasm in live cells, but was found associated with membranes when fixed with formaldehyde in methanol
Summary
Copines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are a family of soluble calcium-dependent membrane-binding proteins [1]. The conservation of copine proteins in single-celled organisms to more complex eukaryotes, suggests copines play fundamental roles in eukaryotic cell processes. Several studies have shown that copines, like other C2 domain containing proteins, are able to bind membranes in a calcium-dependent manner [1,2,3,4,5,6]. Using yeast two-hybrid screening and pulldown experiments, Tomsig et al [10] showed that the A domain of several human copines bind to many different intracellular proteins, which suggests copines have roles in a wide variety of cellular processes
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