A—Topogenesis of membrane proteins at the Sec61 translocon

Secretory and membrane proteins are targeted by hydrophobic signal sequences to the Sec61/SecY translocon for translocation across the membrane or integration into the lipid bilayer. The mechanisms of signal orientation and transmembrane segment integration are only partially understood. The translocon forms a pore across the membrane with a lateral gate for exit of transmembrane segments. It is proposed that membrane integration is the result of thermodynamic equilibration between the pore and the membrane. By mutating Sec61 of yeast, we found that the translocon interior determines the hydrophobicity threshold for membrane integration.

We propose to further explore the interior of the translocon using model substrates, varying the position of hydrophobic residues and sequence geometry. First experiments revealed functional asymmetry within the pore that is dependent on its apolar constriction. We will also analyze the contribution of sequences outside of the trransmembrane segments on stop-transfer or reinsertion activity. In collaboration with Dominic Höpfner (Novartis), we study a new group of fungal dekadepsipeptides that act as potent translocon inhibitors. We expect our experiments to improve our molecular understanding of translocon function and topology determinants.

B—Intracellular protein sorting

Clathrin coated vesicles mediate transport of membrane proteins between the plasma membrane, endosomes, and the trans-Golgi network (TGN). Best characterized are clathrin coats with AP-2 adaptors at the plasma membrane for endocytosis. Much less is known about AP-1/clathrin coats at the TGN and endosomes.

Previous work showed an involvement of AP-1, rab4, and rabaptin-5 in recycling of membrane proteins from endosomes back to the plasma membrane. We propose to analyze their roles in transferrin receptor recycling using automated fluorescence microscopy and in defining endosomes morphology. To study TGN exit, we have developed a tag for the attachment of glycosaminoglycans and discovered that glycan attachment accelerates TGN exit and inhibits endocytosis. We plan to characterize the differences in proteoglycan and nonproteoglycan transport out of the TGN and to isolated transport carriers containing the two forms for mass spectrometric analysis of cargo proteins and components of the sorting machinery.