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 nucleotide-binding domain. For the carboxyl-terminal half (C-half) of Pgp, at least 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 stop-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 investigated the membrane insertion properties of TM segments using the Chinese hamster pgp1 Pgp as a model protein in a cell-free system. We found that TM8 alone or in the presence of TM7 functions as a signal-anchor sequence to insert into membranes with a cytoplasmic amino terminus and an extra-cytoplasmic carboxyl terminus. However, TM8 displayed stop-transfer activity when linked to the C-terminal end of the signal-anchor sequence, TM1. In addition, the membrane orientation of TM8 was found to be regulated by the charge distribution flanking TM8. Interestingly, we found that mammalian and wheat germ ribosomes differentially regulate the signal-anchor and stop-transfer properties of TM8. We conclude that the unique topogenic properties of TM8 direct the generation of multiple C-half topological orientations.
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-0641, USA.
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Zhang
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000502:physiology; D014158:Transcription, Genetic; D014908:Triticum
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals