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YOR1: Plasma membrane ABC transporter protein

     YOR1 encodes an ABC transporter protein that is required for normal resistance to the mitochondrial ATPase inhibitor oligomycin. Yor1p exhibits striking sequence similarity with a group of ABC transporter proteins defined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance protein (MRP). These transporters exhibit a similar predicted topology as shown below.

 

Figure 1. Predicted topology of Yor1p. This structure is proposed based on experimentally verified structures of CFTR and Mrp.

 

Along with the conserved three dimensional structure of these membrane proteins, a great deal of sequence conservation is shared by members of the ABC transporter superfamily. The highest degree of sequence similarity in these ABC transporter proteins is exhibited by the first nucleotide binding domain region (NBD1). These transporters are referred to as the Mrp family of ABC transporters as this close sequence and structural relationship was first recognized during analysis of this protein. An alignment of several Mrp family transporters is shown below.

 

Figure 2. Alignment of NBD1 regions from ABC transporters closely related to Yor1p. The one letter amino acid code is used with important sequences indicated. Walker A and Walker B are motifs found associated with many proteins that bind nucleotides. LSGGQ is a sequence unique to ABC transporters found in their NBDs. DF508 indicates the position of a phenylalanine position that is deleted from ~60% of patients with cystic fibrosis. A mutant form of Yor1p that inserts an alanine residue into the one amino acid gap in the Yor1p NBD1 region is denoted as insA652. Two other members of the Mrp family of transporters are c-MOAT (involved in transport in the liver) and Ycf1p (required for cadmium resistance in S. cerevisiae).

 

From this alignment, it is clear that remarkable sequence conservation is present in these ABC transporters. Our goal is to use Yor1p as a model system for understanding the structure and function of Mrp family members from larger organisms. One important issue central to the biology of Mrp family members is the question of how these proteins arrive at their normal intracellular destinations. The importance of proper intracellular trafficking of Mrp family members is illustrated by the disease cystic fibrosis. 60% of patients with this disease produce a mutant CFTR that contains a single amino acid deletion of phenylalanine 508 in NBD1. Normally, CFTR reaches the plasma membrane of epithelial cells but DF508 CFTR is trapped in the endoplasmic reticulum where it is degraded. Our analysis of the amino acid sequence of Yor1p indicated that this proteins conserved F508 from CFTR as F670 in Yor1p. We constructed a DF670 Yor1p and tested the ability of this protein to complement the oligomycin hypersensitivity of a yor1 mutant strain of yeast.

Figure 3. Mutant forms of Yor1p fail to restore normal oligomycin resistance to a yor1 mutant strain. A strain lacking the YOR1 gene was transformed with low-copy-number plasmids expressing the indicated forms of Yor1p or with a high-copy-number plasmid overproducing DF670 Yor1p (2mm DF670). Transformants were spotted onto medium containing a gradient of oligomycin (increasing as denoted by the bar of increasing width).

 

These complementation data show that the DF670 forms of Yor1p fail to detectably complement the oligomycin hypersensitivity of a yor1 mutant strain. The insertion mutation insA652 surprisingly strongly depressed the ability of the resulting Yor1p derivative to function. Western blot data (not shown) indicated that while the DF670 Yor1p was less stable than wild-type, the insA652 Yor1p was produced at normal steady state levels. The DF508 CFTR is believed to cause disease pathology because the protein cannot normally traffic to the plasma membrane. To determine if DF670 Yor1p also exhibited a localization defect, we compared the subcellular distribution of wild-type, DF670 and insA652 forms of Yor1p through the use of biochemical fractionation. The results of this analysis are shown below.

 

Figure 4. Mutant forms of Yor1p are not normally localized. Protein extracts from S. cerevisiae cells expressing either wild-type, DF670 or insA652 forms of Yor1p were prepared and resolved over 20 to 60% sucrose gradients. Aliquots of each gradient fraction were then electrophoresed on SDS-PAGE and subjected to western blotting using antibodies against Yor1p, the plasma membrane ATPase (Pma1p) or the ER translocon component Sec61p. Top refers to the least dense and bottom refers to most dense sucrose gradient fractions.

As can be seen in figure 4, loss of F670 from Yor1p leads to the protein shifting from being enriched in the most dense gradient fractions to localization to the center of the gradient. This distribution closely parallels that of the ER marker, Sec61p. Note that insA652 Yor1p exhibits a biphasic distribution with a small amount of the protein reaching the plasma membrane while the majority of the protein is found in the ER-enriched fractions. These fractionation profiles closely correlate with the resistance phenotypes as only the Yor1p derivatives that can reach the plasma membrane show detectable oligomycin resistance.

Our goal is to use the subcellular trafficking of Yor1p in S. cerevisiae as a model for delivery of ABC transporters like CFTR and Mrp to their normal residences in mammalian cells. The easily selectable oligomycin resistance phenotype provided by yor1 mutants will be exploited to use genetics to examine this issue. We hope to identify proteins involved in trafficking of Yor1p that have homologues in animal cells.

 


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December 10, 1999.