Structure/function analysis of the Mrs2p

Mg2+ transport proteins of eukaryotes (Mrs2p, Alr1p) and of prokaryotes (CorA) for a large, diverse superfamily. While other laboratories recently solved the CorA 3-D structure, we have characterized Mrs2p as a Mg2+-specific ion channel. Now we aim at performing a comparative study on structure and function of Mrs2 and CorA proteins to unravel similarities and differences. To this end three laboratories combine their competence in X-ray crystallography, in electrophysiology and in genetics. Due to the particular chemistry of the Mg2+ ion and the unique structure of the CorA-Mrs2-Alr1 protein family we expect to obtain new insights into function and regulation of an ion channel and its role in cellular physiology.

Physiological studies of several decades described various activities of uptake and extrusion of ions across the mitochondrial inner membrane. Cations like K+ , Na+ , Ca2+ or Mg 2+ have been found to be taken up by electrophoretic processes, making use of high, negative membrane potential inside organelles as a driving force. A genetic screen in the yeast Saccharomyces cerevisiae resulted in the identification of MRS2 encoding a protein mediating Mg 2+ uptake into mitochondria. It is a distant homologue of the ubiquitous bacterial CorA Mg 2+ transport protein and of the yeast plasma membrane Mg 2+ transport protein Alr1. Mammalian genomes express a single functional orthologue of yMrs2p in their mitochondria, fungal genomes encode two orthologues (Mrs2p and Lpe10p in yeast). Members of the CorA-Mrs2-Alr1 superfamily thus appear to be the predominant, major Mg 2+ transport proteins in bacteria, lower eukaryotes and plants, whereas mammals make use of a single member of the Mrs2 family in their mitochondria only. In common to all members of the CorA-Mrs2-Alr1 protein superfamily are two adjacent transmembrane helices (TM-A, TM-B) near their C- terminus. TM-A terminates with a F/Y-G-M-N motif. The yeast Mrs2 protein exposes both the long N-terminal and the shorter C-terminal sequences towards the mitochondrial inside (matrix). Only the short sequence connecting the TM domains appears on the outer side of the inner mitochondrial membrane (intermembrane space). This project aimed at a combined structural and physiological approach to study CorA and Mrs2 proteins to develop a detailed picture of this unique family of channel proteins and their mechanism of Mg 2+ conductance. Being the first characterized channel protein of the inner mitochondrial membrane, Mrs2 can serve as a model for ion uptake into this organelle and patho-pyhsiological effects of its malfunction. Our studies provide the first molecular view of the regulatory N-terminal moiety of the eukaryotic magnesium transporter Mrs2 from S. cerevisiae. Together with structure-based mutagenesis and functional analysis in vivo, we obtained insight into gating and regulation of Mrs2 Mg 2+ transport. We identified candidate residues implicated in divalent cation binding, residues involved in formation of the hydrophobic gate, and showed that the entire C- terminal region acts as the basic sphincter. The highly conserved G-M-N motif of the CorA-Mrs2-Alr1 family of Mg 2+ transporters has been proven to be essential for Mg 2+ transport. We performed random mutagenesis of the G-M-N motif of Saccharomyces cerevisiae Mrs2p, and a genetic screen. We obtained a large number of mutants still capable of Mg 2+ transport, albeit below the wild-type level, as assessed by measurements of Mg 2+ influx into isolated mitochondria. Further, we systematically analysed the conserved hydrophobic gate region of the magnesium transporter CorA and confirmed the importance of Leu294 in gating. In addition, the Mrs2 homologue Lpe10p was shown to modulate the activity of the Mrs2p-based yeast mitochondrial Mg 2+ channel, while lacking transport function in the absence of Mrs2p. In summary, our studies contributed to the molecular view on Mg 2+ transport systems by elucidating permeation properties together with regulatory gating processes.