Determinants of membrane protein topology

The living cell, no matter if it is a simple prokaryotic bacterial cell or a highly specialised eukaryotic cell, synthesises a vast number of different proteins. In most organisms, 20-30 % of all proteins are thought to be transmembrane integral membrane proteins. Membrane proteins can be classified into several subgroups, depending on their mode of interaction with the membrane. There are two major types of membrane proteins, integral and peripheral. Peripheral membrane proteins are bound to the surface of the membrane, interacting with other membrane proteins or with one leaflet of the bilayer. The other group, integral membrane proteins, can be subdivided into two major types, transmembrane and membrane-anchored. Transmembrane proteins are integrated into the membrane via internal hydrophobic amino acid sequences, while membrane-anchored proteins are attached to the membrane via covalently bound lipids or so-called GPI anchors. This thesis is based on studies of the rules governing the biogenesis of transmembrane integral membrane proteins. The influence of positively charged amino acids on protein translocation has been compared in Escherichia coli and in a mammalian system. The influence of positively and negatively charged amino acids on the conformation of a transmembrane helix in the ER membrane has been studied. Topology mapping of two Escherichia coli proteins has provided the first known example of divergent evolution of membrane protein topology. Finally, the topology of the Escherichia coli potassium channel Kch has been determined.