Reorientation of aquaporin-1 topology during maturation in the endoplasmicreticulum

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coo rdinated translocation and membrane integration events. For the human aquap orin water channel AQP1, however, the initial four-segment-spanning topolog y at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follo w the transmembrane (TM) orientation of key internal peptide loops in Xenop us oocyte and cell-free systems. This analysis revealed that AQP1 maturatio n in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underw ent a 180-degree rotation in which TM3 C-terminal flanking residues were tr anslocated from their initial cytosolic location into the ER lumen and N-te rminal flanking residues underwent retrograde translocation from the ER lum en to the cytosol. These events convert TM3 from a type I to a type II topo logy and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological re orientation was also associated with maturation from a protease-sensitive c onformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cot ranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.