COUPLED TRANSLOCATION EVENTS GENERATE TOPOLOGICAL HETEROGENEITY AT THE ENDOPLASMIC-RETICULUM MEMBRANE

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we sh ow, however, that through the coupling of sequential translocation eve nts, native topogenic determinants are capable of generating two alter nate transmembrane structures at the endoplasmic reticulum membrane. U sing defined chimeric and epitope-tagged full-length proteins, we foun d that topogenic activities of two C-trans (type II) signal anchor seq uences, encoded within the seventh and eighth transmembrane (TM) segme nts of human P-glycoprotein were directly coupled by an inefficient st op transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a sin gle- and a double-spanning TM topology with nearly equal efficiency. I n addition, ST and C-trans signal anchor activities encoded by TM8 wer e tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features cont ributing to this unusual topogenic behavior included 1) a short, flexi ble peptide loop connecting TM7a and TM7b, 2) hydrophobic residues wit hin TM7b, and 3) hydrophilic residues between TM7b and TM8.