Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening.

A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-beta-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others

Structural basis for alternating access of a eukaryotic calcium/proton exchanger

Eukaryotic Ca(2+) regulation involves sequestration into intracellular organelles, and expeditious Ca(2+) release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal of Ca(2+) after such signalling events is accomplished by members of the Ca(2+):cation (CaCA) superfamily. The CaCA superfamily includes the Na(+)/Ca(2+) (NCX) and Ca(2+)/H(+) (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca(2+) resting potential in muscle cells, neuronal signalling and Ca(2+) reabsorption in the kidney. The CAX family members maintain cytosolic Ca(2+) homeostasis in plants and fungi during steep rises in intracellular Ca(2+) due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones. The cytosol-facing conformations within the CaCA superfamily are unknown, and the transport mechanism remains speculative. Here we determine a crystal structure of the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger (Vcx1) at 2.3 Å resolution in a cytosol-facing, substrate-bound conformation. Vcx1 is the first structure, to our knowledge, within the CAX family, and it describes the key cytosol-facing conformation of the CaCA superfamily, providing the structural basis for a novel alternating access mechanism by which the CaCA superfamily performs high-throughput Ca(2+) transport across membranes