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Synthesis and Maturation of Oligomeric Membrane Proteins

M.M. Falk

My laboratory is studying the mechanisms and processes involved in the synthesis, assembly, targeting, and degradation of oligomeric membrane proteins. Polytopic membrane protein subunits of biological channels are of particular interest. These subunits are specialized membrane proteins that bear charged amino acid residues within the transmembrane domains to allow formation of hydrophilic pores within the lipid bilayer.

Recently, my colleagues and I investigated synthesis of connexin polypeptides and assembly of the connexins into gap junction channels. Gap junctions are transmembrane, double-membrane channel structures that combine the cytoplasms of 2 adjacent cells (Fig. 1). Two half-channels or connexons, which are similar in structure to ion channels, pair via the extracellular domains of the connexin subunits to form the complete gap junction channel. Connexins in several ways are similar to other channel-forming membrane proteins. However, some untypical features associated with their unique function make them interesting biological objects.

Using cell-free translation in the presence of membrane vesicles derived from the endoplasmic reticulum, we showed that connexins are cotranslationally integrated into the membrane of the endoplasmic reticulum in a reaction that requires "signal recognition particle." N-glycosylation site tagging and protease protection assays revealed that the functional transmembrane topology of connexins is achieved during integration of the proteins into the membrane of the endoplasmic reticulum. Optimization of the cell-free expression system resulted in efficient assembly of functional gap junction connexons (Fig. 2), indicating that assembly of connexins into hexameric connexons is achieved in the membranes of the endoplasmic reticulum. This result is consistent with assembly in the endoplasmic reticulum, as described for other oligomeric proteins. Using subcellular fractionation and immunocolocalization, we obtained strong evidence that connexins are transported from their site of synthesis in the endoplasmic reticulum through the Golgi stacks to their site of function in the plasma membrane, following the common intracellular transport route referred to as the secretory pathway.

Recently, we tagged several connexin isotypes with green fluorescent protein. The tagged connexins were synthesized and trafficked efficiently to the plasma membrane, and they assembled into functional gap junction channels. Current research has 3 primary aims: (1) investigate insertion and positioning of the nascent connexin polypeptides within the protein-translocating channel located in the membrane of the endoplasmic reticulum, (2) characterize signals within the connexin sequences that regulate assembly into homo-oligomeric and hetero-oligomeric connexons, and (3) use the connexins tagged with green fluorescent protein to obtain a detailed picture of the steps involved in the synthesis, formation, and degradation of gap junction channels in living cells.

PUBLICATIONS

Falk, M.M., Buehler, L.K., Kumar, N.M., Gilula, N.B. Cell-free expression of functional gap junction channels. In: Gap Junctions. Werner, R. (Ed.). IOS Press, Washington DC, 1998, p. 135.

Falk, M.M., Buehler, L.K., Kumar, N.M., Gilula, N.B. Cell-free synthesis and assembly of connexins into functional gap junction membrane channels. EMBO J. 16:2703, 1997.

Falk, M.M., Gilula, N.B. Connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access. J. Biol. Chem. 273:7856, 1998.

Kahn, T.W., Beachy, R.N., Falk, M.M. Cell-free expression of a GFP-fusion protein allows protein quantitation in vitro and in vivo. Curr. Biol. 7:R207, 1997.

Yeager, M., Unger, V., Falk, M.M. Synthesis, assembly, and structure of gap junction intercellular channels. Curr. Opin. Struct. Biol. 8:517, 1998.