Functional expression of human mdr1 in the yeast Saccharomyces cerevisiae.
AUTOR(ES)
Kuchler, K
RESUMO
Development of multiple drug resistance in tumor cells involves amplification of the mdr1 gene product, a 170-kDa plasma membrane glycoprotein that is an ATP-driven pump that extrudes the drugs. Human mdr1 (also designated as PGY1) cDNA was expressed in yeast cells by using the promoter and translational initiation signal of a related yeast gene, STE6. Immunoblotting of subcellular fractions showed that all of the Mdr1 (also known as P glycoprotein) was associated with the particulate material. Immunofluorescence microscopy revealed that the majority of the Mdr1 was localized to the plasma membrane (although a significant amount was also found in the endoplasmic reticulum). In contrast to mammalian cells, Mdr1 was not glycosylated in yeast. Nevertheless, some, if not all, of the Mdr1 made in yeast was properly folded and functional because it could be photoaffinity labeled specifically with 8-azido-ATP and because cells overexpressing Mdr1 displayed increased resistance towards valinomycin, an ionophore known to interact with Mdr1 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=48645Documentos Relacionados
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