Co-translational effects of temperature on membrane insertion and orientation of P-glycoprotein sequences

P-glycoprotein (pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter, a volume-regulated chloride channel, and an ATP channel. Previously, it has been shown that hamster pgp 1 Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 and by soluble cytoplasmic factors. Different topological structures of Pgp may be related to its different functions. In this study, we examined the effects of translation temperature on the membrane insertion process and the topologies of Pgp. Using the rabbit reticulocyte lysate expression system, we showed that translation at different temperatures affects the membrane insertion and orientation of the putative TM3 and TM4 of hamster pgp 1 Pgp in a co-translational manner. This observation suggests that the membrane insertion process of TM3 and TM4 of Pgp molecules may involve a protein conducting channel and/or the interaction between TM3 and TM4, which act in a temperature sensitive manner. We speculate that manipulating temperature may provide a way to understand the structure-function relationship of Pgp and help overcome Pgp-related multidrug resistance of cancer cells.