Abstract
The pleiotropic drug resistance protein, Pdr5p, is an ATP-binding cassette transporter of the plasma membrane of Saccharomyces cerevisiae. Overexpression of Pdr5p results in increased cell resistance to a variety of cytotoxic compounds, a phenotype reminiscent of the multiple drug resistance seen in tumor cells. Pdr5p and two other yeast ATP-binding cassette transporters, Snq2p and Yor1p, were found to be phosphorylated on serine residues in vitro. Mutations in the plasma membrane-bound casein kinase I isoforms, Yck1p and Yck2p, abolished Pdr5p phosphorylation and modified the multiple drug resistance profile. We showed Pdr5p to be ubiquitylated when overexpressed. However, instability of Pdr5p was only seen in Yck1p- and Yck2p-deficient strains, in which it was degraded in the vacuole via a Pep4p-dependent mechanism. Our results suggest that casein kinase I activity is required for membrane trafficking of Pdr5p to the cell surface. In the absence of functional Yck1p and Yck2p, Pdr5p is transported to the vacuole for degradation.
Highlights
Overexpression of human P-glycoprotein MDR1 is responsible for the phenomenon of multiple drug resistance in various resistant cell lines and tumors
The 100-kDa polypeptide was the plasma membrane Hϩ-ATPase, Pma1p, previously shown to be phosphorylated by casein kinase I [48], whereas the 160-kDa band consisted of Pdr5p, Snq2p, and Yor1p, as the signal intensity was drastically decreased in the PDR1 wildtype IL125-2B strain, which does not overproduce these three ATP-binding cassette (ABC) proteins, and successive deletions of PDR5, SNQ2, and YOR1 in the US50 –18C strain resulted in a decreasing intensity of the 160-kDa signal (Fig. 1A)
We examined the phosphorylation of plasma membranes prepared from the SUPERYOR strain [43], which contains multiple deletions of the ABC transporter genes, including SNQ2 and PDR5, and expresses YOR1 from the PDR5 promoter (PPDR5::YOR1)
Summary
Overexpression of human P-glycoprotein MDR1 is responsible for the phenomenon of multiple drug resistance in various resistant cell lines and tumors. P-glycoprotein is an integral plasma membrane protein that acts as an ATP-dependent efflux pump to extrude seemingly unrelated hydrophobic compounds from the cell interior, conferring resistance to a large variety of drugs [1, 2]. It belongs to the ATP-binding cassette (ABC) transporter superfamily and consists of a tandem repeat of transmembrane domains and conserved nucleotide-binding motifs, connected by a central linker region [1, 3, 4]. Elevated levels of Pdr5p and Snq2p lead to growth resistance to a distinct subset of structurally unrelated xenobiotics (16 –21). The replacement of serine residues by alanine in a PEST-like sequence of Fur4p [33] and in a sequence surrounding a ubiquitination site of Ste2p [34] decreases the level of phosphorylation and ubiquitination of each protein
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