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Biosynthesis of Oligomeric Membrane Proteins

M.M. Falk, P. Shen

Biological membranes play a key role in life. Membranes not only surround cells but also divide eukaryotic cells into subcellular compartments, allowing the formationof different environments within a cell. The membrane lipids, however, form a tight seal and prevent simple exchange of biological molecules. We are interested in understanding what role membrane proteins might play in transmembrane transport processes.

Polytopic membrane protein subunits of biological channels are of particular interest. These subunits are specialized membrane proteins that have charged amino acid residues within the transmembrane domains to allow formation of hydrophilic pores within the lipid bilayer. Oligomeric gap junctions are an example of such biological channels. They cluster in large plaques in the adjoining plasma membranes of neighboring cells and provide direct cell-to-cell communication. Some untypical features associated with their unique function are extremely interesting.

We showed that connexins are cotranslationally integrated into the endoplasmic reticulum, where they achieve their functional transmembrane topology, and subsequently are transported through the Golgi apparatus to reach the plasma membrane. Investigating the assembly of gap junction channel subunits in a cell-free translation system, we found that different connexin isotypes interact selectively by allowing the assembly of only homo-oligomeric and certain types of hetero-oligomeric channels. The results also suggest that both "assembly" signals that regulate principal recognition of connexin subunits and "selectivity" signals that regulate specific assembly of hetero-oligomeric channels may exist in the connexin polypeptide sequence.

To study the biosynthesis of gap junctions in live cells, we tagged connexins with green fluorescent protein (GFP; Fig. 1). The connexin-GFP fusion proteins were efficiently expressed, oligomerized, and transported to the plasma membrane, where they assembled into functional gap junction channels and gap junction plaques (Fig. 2). Deconvolved, high-resolution fluorescence images of gap junction plaques assembled from the subunit a₁Cx43 tagged with GFP revealed a rich plaque morphology in both 2- and 3-dimensional image reconstruction.

Time-lapse images showed that large gap junction plaques are formed by the fusion of smaller plaques and that gap junction plaques are not rigid structures but are extremely dynamic in appearance and shape. These dynamic images of live cells add a new dimension to the classical static picture of gap junctions and provoke a number of interesting questions about the structure and function of the junctions.

Our current research has 3 primary aims: (1) to characterize chaperones and other factors that play a role in the biosynthesis of gap junction channels, (2) to further characterize the signals that regulate the assembly of gap junction channel subunits, and (3) to use the GFP-tagged connexins in combination with high-resolution, multidimensional fluorescence deconvolution microscopy to investigate the biosynthesis of gap junction channels in live cells.

PUBLICATIONS

Falk, M.M. Cell-free expression for analyzing the membrane integration, oligomerization, and assembly characteristics of gap junction connexins. Methods, in press.

Falk, M.M. Connexins/connexons: Cell-free expression. Methods Mol. Biol., in press.