PLASMA MEMBRANE DOMAINS: PHOTOSYNTHESIS, IONS, PROTEOMICS

Charasomes are convoluted plasma membrane domains in internodal cells of the characean green alga, Chara australis. According to the current hypotheses, hitherto based on indirect evidence, the charasomes are responsible for proton extrusion, required for effective carbon acquisition and photosynthesis, and play an important role in Cl- uptake and Ca2+ transport. So far nothing is known about the protein composition of charasomes apart from an uncharacterized proteoglucan which may be responsible for membrane curvature. We found recently that charasomes can be stained in vivo by fluorescent dyes allowing a direct comparison with membrane transport processes and properties. During the course of this project we want to verify or discard the following hypotheses: (1) Charasomes are involved in local proton extrusion under steady state conditions. (2) Charasome- rich areas are regions of enhanced photosynthesis in undisturbed branchlet internodal cells. (3) Charasomes are involved in Cl- uptake but not in active Ca2+ transport. (4) The presence of charasomes affects intracellular ion concentration and subcellular plasma membrane potential. (5) Charasomes are characterized by a specific proteoglycan but not by charasome-specific transporters. For our study we combine electrophysiological methods with fluorescence and confocal laser scanning microscopy. We will apply mass spectrometry analysis in order to identify proteins specifically enriched in or absent from charasomes in comparison with smooth plasma membrane areas. The research proposed in this application will extend our knowledge about the role of lateral organization of biological membranes in transmembrane transport.

Plant cells specialized on transport of ions and metabolites form elaborate plasma membrane infoldings in order to accommodate transporters. A convenient model to study complex plasma membrane areas are the internodal cells of the characean green algae. Their "charasomes" are involved in the local acidification of the external medium, which facilitates efficient uptake of carbon required for photosynthesis. Along the cell surface, the acidic regions alternate with alkaline zones required for charge balance. During the course of the project, we applied molecular biology, subcellular proteomics and imaging methods with high spatial and temporal resolution in order to identify transporters and other proteins, to reveal their differential distribution along the cell surface and to investigate their role the turnover of charasomes. We found that stable acid regions with a high abundance of charasomes were enriched in H+ ATPases (transporters required for acidification) not only because of the increased membrane surface but also because of a denser packing. The sequence of the Chara plasma membrane H+ ATPase was analyzed and a putative OH- transporter responsible for local alkalinization was discovered. We found further that the activation/deactivation of Chara plasma membrane transporters and the long-distance communication between anchored chloroplasts could be modified by inhibitors, which interfered with organelle trafficking. This showed unexpected interactions between distantly related metabolic pathways. Our findings also revealed new aspects of charasome turnover. Light-induced formation of charasomes required local inhibition of specific enzymes necessary for clathrin-dependent membrane recycling and this inhibition was released upon dark incubation. Clathrin proteins were also enriched at non-degrading charasomes indicating an important role in the architecture of these 3D domains. In the light, charasome degradation could be induced when internodal cells were aligned parallel to each other. This led to matching of pH patterns, the photosynthetic activity of chloroplasts and, after several days, to altered charasome patterns. Thereby we showed for the first time that plant cells interact with each other via differences in surface pH.