[CANCER RESEARCH 54, 3202-3209, June 15, 1994J ABSTRACT Understanding how the multidrug resistance phenotype is manifest in human cancer cells will require inslajat into the mechanism of assembly, transmembrane topology, and Intracellular trafficking ofhuman P-glyco protein (MDR1). Previously, we showed that MDR1 amino terminus biogenesis occurred through an unexpected Interaction between novel topogenic sequence subtypes and that transmembrane topology of corre spending amino and carboxy halves was not equivalent. We now investi gate topology and topogenic activities of the third and fourth transmem brane regions (TM3 and TM4) ofhuman MDR1 using protease protection of defined reporter epitopes expressed in Xenopus laevis oocytes. As was previously observed for TM1 and TM2, determinants in TM3 and TM4 exhibited cooperativity in directing proper assembly and transmembrane orientation. The signal sequence encompassing TM3 required residues from TM4 to reinitlate translocation of the MDR1 chain into the endo plasmic reticulum (ER) lumen. Remaining residues from TM4 terminated translocation and established a polytopic transmembrane topology in which TM3 and TM4 both spanned the membrane in the orientation predicted by hydropathy-based models. Remarkably, when translocating sequentially into the ER lumen, neither TM4 alone nor TM4 together with TM3 efficiently terminated translocation. Thus, MDR1 biogenesis me quired both the presence of these sequences and their proper orientation with reaped to the ER translocation apparatus. This conclusion was supported by experiments in which TM3 and TM4 topology was repro duced in a defined chimeric protein which mimicked native MDR1 pro sentation. These additional variations on simple themes of protein tape genesis utilized by MDR1 demonstrate that events of complex protein biogenesis may be dissected and studied using protein chimeras with defined traaslocation properties. INTRODUCTION Transmembrane topology of most eukaryotic polytopic IMPs3 is established at the ER membrane coincident with protein synthesis (1—4). One model for generating polytopic topology proposes that independent topogenic sequences with properties similar to S se quences, ST, or SA sequences direct sequential translocation and membrane integration events through interactions with receptor pro teins at the ER membrane (5—7).Support for this model comes from two types of studies: those in which IMPs of predicted polytopic topology were generated by expression of chimeras encoding S. ST, and/or SA sequences (e.g., S-ST-S, SA-ST-SA, and SA-SA-SA) (4, 8, 9)andotherstudiesin which initial topogenicsequencesof naturally occurring polytopic proteins such as bovine rhodopsin, uracil per mease, and the acetylcholine receptor were found to be functionally indistinguishable from conventional 5, SA, or ST sequences (10—12). Received 12/6/93; accepted 4120/94. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from the National Cystic Fibrosis Foundation to V. L. and a Physician Scientist award (CA01614) to W. S. 2 To whom requests for reprints should be addressed, at Department of Physiology, University of Califomia, San Francisco, San Francisco, CA 94143-0444. 3 The abbreviations used are: IMP, integral membrane protein; ABC protein, AlP binding cassette protein; ER, endoplasmic reticulum; MDR1, human multidrug resistance protein, P-glycoprotein; piçproteinase K SDS, sodium dodecyl sulfate; S, signal; SA, signal anchor; ST, stop transfer; TM, transmembrane region; cDNA, complementary DNA; PCR, polymerase chain reaction; XO, Xenopus lacy/s oocytcs. Human P-glycoprotein, MDR1, is a polytopic integral membrane protein which exports a diverse group of cytotoxic drugs from human cells (13—15).It is also a member of the ABC protein superfamily which performs a wide variety of cellular transport functions (16, 17). It was previously thought, based on hydropathy analysis and homol ogy with bacterial hemolysin B, that MDR1 and eukaryotic relatives [i.e., CVFR (cystic fibrosis transmembrane conductance regulator) and STE 6 (sterile 6)] exhibited a tandem repeat structure with each half of the molecule containing six hydrophobic membrane-spanning helices and a large cytosolic AlP-binding domain (18—21).Recent studies have called into question not only this tandem repeat topolog ical model (22—24)but also current hypotheses concerning how the ABC transport superfamily is assembled into membranes (25). In vivo studies of reporter-protein chimeras and in vitro studies of both full-length and truncated human MDR1 have demonstrated that the carboxy terminus spans the membrane four rather than six times as predicted by hydropathy (22). Topology of the first and second verses the seventh and eighth putative transmembrane helices (TM1-TM2 versus TM7-TM8, respectively) demonstrated an asymmetric rather than symmetric topology for homologous halves of the protein (25). In addition, initial events in MDR1 topogenesis exhibited an unexpected complexity in that they were not directed by independent SA or ST sequences (25). Instead, two independent topogenic sequences (TM1 and TM2, respectively) functioned in concert to establish integration of the nascent chain into the lipid bilayer. Studies by Zhang and Ling (23) using cell-free analysis of truncated murine P-glycoprotein also demonstrated a carboxy terminus topology which spanned the mem brane only four times. Similar in vitro studies of hamster P-glyco protein demonstrated two alternate topologies for the amino terminus, one which spanned the membrane six times and a second which spanned only four times (24). To further define the mechanism of biogenesis of human MDR1, we have used a Xenopus oocyte expression system (26) to examine topology of polypeptides generated from cDNA clones containing a reporter of translocation engineered into sequential sites of the MDR1-coding region. With this technique we confirmed the previ ously reported transmembrane topology of TM1 and TM2 (25) and demonstrated that TM3 and TM4 (in contrast to TM9 and TM1O) spanned the membrane in the orientation predicted by hydropathy based models. An internal signal sequence encompassing TM3 and a significant portion of TM4 (residues 139—226)reinitiated transloca tion of the MDR1 nascent chain. Although TM4 along with its flanking sequences terminated translocation when it was preceded by TM3 in its native context, TM4 lacked efficient de novo stop transfer activity. Finally, native topology of TM4 and/or TM3 was reconsti tuted in a complex protein chimera, demonstrating the requirement for both the presence as well as proper orientation of these sequences with respect to the ER translocation apparatus. MATERIALS AND METHODS Materials. DNA-modifying enzymes were purchased from New England Biolabs. Oligonucleotides were synthesized at the University of California, San Farancisco, Biomedical Resource Center. Tran35S-label was purchased from ICN Radiochemicals, and proteinase K was purchased from Boehringer 3202 Transmembrane Orientation and Topogenesis of the Third and Fourth Membrane-spanning Regions of Human P-Glycoprotein (MDR1)' William R. Skach and Vishwanath R. Lingappa2 Departments of Medicine [W. R. S., V. R. L.J and Physiology [V. R. L.J and Cancer Research Institute [W. R. S.J, University of California, San Francisco, San Francisco, California 94143-0444 Research. on September 28, 2021. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from