MECHANISM INVOLVED IN GENERATING THE CARBOXYL-TERMINAL HALF TOPOLOGY OF P-GLYCOPROTEIN

P-Glycoprotein (Pgp) is a polytopic membrane protein that consists of a tandem repeat of a transmembrane (TM) domain followed by a nucleotid e-binding domain. For the carboxyl-terminal half (C-half) of Pgp, at l east three different topological orientations have been observed. One major difference between these topologies is reflected in the membrane insertion property of TM8, which is predicted to (1) function as a st op-transfer sequence, (2) lack stop-transfer activity, or (3) function as a signal-anchor sequence. To understand the mechanism involved in generating multiple topological forms for the C-half of Pgp, we invest igated the membrane insertion properties of TM segments using the Chin ese hamster pgpl Pgp as a model protein in a cell-free system. We foun d that TM8 alone or in the presence of TM7 functions as a signal-ancho r sequence to insert into membranes with a cytoplasmic amino terminus and an extra-cytoplasmic carboxyl terminus. However, TM8 displayed sto p-transfer activity when linked to the C-terminal end of the signal-an chor sequence, TM1. In addition, the membrane orientation of TM8 was f ound to be regulated by the charge distribution flanking TM8. Interest ingly, we found that mammalian and wheat germ ribosomes differentially regulate the signal-anchor and stop-transfer properties of TM8, We co nclude that the unique topogenic properties of TM8 direct the generati on of multiple C-half topological orientations.