|Functional Expression of Human mdr1 in the Yeast Saccharomyces cerevisiae|
Kuchler K, Thorner J
Proceedings of National Academy of Sciences USA (1992)
Category: mitochondria, potassium, yeast-misc ¤ Added: Mar 13, 2009 ¤ Rating: ◊◊
Development of multiple drug resistance in tumor cells involves amplification of the mdrl gene product, a 170-kDa plasma membrane glycoprotein that is an ATP-driven pump that extrudes the drugs. Human mdrl (also designated as PGYI) cDNA was expressed in yeast cells by using the promoter and translational initiation signal ofa related yeast gene, STE6. Immunoblotting of subcellular fractions showed that all of the Mdrl (also known as P glycoprotein) was associated with the particulate material. Immunofluorescence microscopy revealed that the maijority ofthe Mdrl was localized to the plasma membrane (although a signifcant amount was also found in the endoplasmic reticulum). In contrast to mammalian cells, Mdrl was not glycosylated in yeast. Nevertheless, some, if not all, of the Mdrl made in yeast was properly folded and functional because it could be photoaffinity labeled specifically with 8-azido-ATP and because cells overexpressing Mdrl displayed increased resistance towards valinomycin, an ionophore known to interact with Mdrl in animal cells. Hence, a human polytopic membrane protein was correctly inserted into the yeast plasma membrane, and glycosylation was not required for its function.