INTRODUCTION

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Multiple-drug resistance has often been linked with increased expression of ATP-binding cassette (ABC) transporter proteins that act as multispecific drug efflux pumps (see reference 15 for a review). The first known ABC transporter protein mediating multidrug resistance was human MDR1. MDR1 is localized to the plasma membranes of cells and is overexpressed in an array of multidrug-resistant cell lines and tumors (reviewed in reference 57). Biochemical experiments indicate that MDR1 transports compounds that typically have not been modified by the cell (reviewed in reference 16).

More recently, a second ABC transporter has been found in multidrug-resistant lung tumor cells (8). This ABC transporter protein, designated multidrug resistance protein (MRP), has several properties that make it distinct from MDR1. First, MRP transports modified substrates, such as glutathione and glucuronide conjugates (28, 36, 39, 45). Second, while both MDR1 and MRP possess a repeating structure of a set of transmembrane domains followed by the characteristic ABC transporter nucleotide binding domain, MRP has an additional set of transmembrane domains at its amino terminus (2). Finally, nucleotide binding domain 1 (NBD1) of MRP exhibits a characteristic spacing of functional motifs and high sequence similarity that serves to define a group of ABC transporter proteins referred to as the MRP family (8).

Like all known ABC transporters, NBD1 of the MRP family contains a Walker A, LSGGQ, and Walker B element (26, 64). The identical spacing of these elements in NBD1 of the MRP family serves to define this class of ABC transporter proteins (8). A second conserved feature in the MRP family NBD1 region is the presence of a phenylalanine residue between the Walker A and LSGGQ motifs. This phenylalanine was shown to be precisely deleted from an MRP family member, the human cystic fibrosis (CF) transmembrane conductance regulator (CFTR), in 60% of patients with CF (62). Wild-type CFTR must arrive at the plasma membrane to function normally, and deletion of this phenylalanine residue (F508) causes the resulting mutant protein to be retained in the endoplasmic reticulum (ER) (7), where it is degraded by the proteasome (29, 65).

Saccharomyces cerevisiae has also been found to contain a group of ABC transporter proteins showing the characteristic structural features indicative of the MRP family. Ycf1p was the first member of the S. cerevisiae MRP family and was identified by its important role in cadmium tolerance (60). A second member of the S. cerevisiae MRP family was cloned as a key determinant of oligomycin resistance (9, 33). This ABC transporter protein was designated Yor1p (stands for yeast oligomycin resistance protein).

In this work, we localize Yor1p to the plasma membrane in cells. Analyses of wild-type and mutant forms of Yor1p suggest that the trafficking and turnover of this yeast protein have remarkable similarity to those of human CFTR. As seen for the major disease-associated allele of CFTR (F508), degradation of an analogous form of Yor1p involves multiple proteolytic systems.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast strains and media. The S. cerevisiae strains used are listed in Table 1. Yeast transformation was performed by the lithium acetate method as described previously (27). Standard yeast media containing supplements appropriate for growth of auxotrophic strains (56) were employed for growth of cells. Selection with the drug oligomycin was performed as described previously (33). Gradient plates were prepared by pouring 25 ml of YPGE medium (56) containing the desired maximum concentration of oligomycin into plates resting at an incline. Once this drug-containing medium had solidified, plates were laid flat and overlaid with YPGE medium (25 ml). Relative resistance levels were then assessed by performing a spot test assay (69) along the gradient.

Plasmids. The low-copy-number plasmid containing the YOR1 gene was constructed in several steps. First, a 1.4-kb SacII/SnaBI fragment from YOR1 was cloned into pBluescript KSII() (cut with SacII and EcoRV). The HindIII site that lies 3' to the insert was then removed by cleaving the sequence with HindIII and XhoI and treating it with the Klenow fragment and deoxynucleoside triphosphates, followed by religation to make plasmid pDK42. The SacII/KpnI fragment from pDK42 was then cloned into pRS316 that had been cut with the same enzymes, resulting in plasmid pDK58. The 5' end of the gene was then cloned into pDK58 as a SacI/SacII fragment, giving rise to the full-length gene in pRS316 (pDK59). The template for site-directed mutagenesis (pSM111) contained the HindIII/SpeI fragment from nucleotides +1826 to +2913 relative to translation start site (which contains the first nucleotide binding domain) in plasmid pBCSK+ (Stratagene).

Site-directed mutagenesis. Mutations in the first nucleotide binding domain of YOR1 were introduced by a PCR-based strategy (54). Briefly, mutagenic primers were annealed to the pSM111 template along with the T7 primer. The mutagenic primers were as follows: F670, TCT TTA TTG AAT GGg GAt CC TAT GAT GTT ATC TCT TAC AG; K715M, CCT GGC TAA ATT GAT tCt gGC CaT TTG ACC ACC AGA TAA AGT AAT ACC; K715Q, CCT GGC TAA ATT GAT ACG TGC ttg TTG ACC ACC AGA TAA AGT AAT ACC; and insA652 (alanine insert at position 652), CCA TGG ATA ACC ACA CAT TAA agc TAA GTC CCC GTT GAC TTC AAC C. Nucleotides that differ from those of the wild type are indicated in lowercase letters. A second PCR used a YOR1 primer (+2090 to +2110) and the T3 primer. The products of each of the mutagenic reactions and the overlapping product were gel purified, combined, and used for a second PCR with the T3 and T7 primers. The products of the second PCR were cloned back into pSM111 as either a HindIII/Spe1 fragment (in the case of the alanine insertion) or an Nsi1/SpeI fragment (with the K715M, K715Q, and F670 mutations). Following verification of mutations by restriction mapping, plasmids were sequenced to ensure that no additional errors had been introduced. DNA fragments containing the mutations were then placed in the context of the wild-type gene.

Production of rabbit anti-Yor1p antiserum. Antisera directed against either the amino-terminal 110 amino acids or the 80 carboxy-terminal amino acids were used in this study. The production of the C-terminal antiserum has been described elsewhere (33). A fusion protein between glutathione S-transferase (GST) and the amino-terminal 110 amino acids of Yor1p was constructed in plasmid pGEX-KG (17). PCR was used to amplify the region and introduce EcoRI and HindIII sites at amino acids 1 and 110, respectively. This allowed the fragment to be cloned into the expression vector cleaved at these same sites. The resulting plasmid was designated pDK40 and sequenced to ensure that no errors had been introduced. The GST-Yor1p fusion protein was purified by standard methods and used to immunize rabbits as described previously (20, 33). Both the N-terminal and C-terminal antisera were affinity purified with Aminolink Plus (Pierce) matrix coupled to appropriate GST-Yor1p fusion proteins by the manufacturer's protocol.

Immunoblotting. Cells were grown in complete synthetic medium minus uracil to an optical density at 600 nm (OD₆₀₀) of 0.3 to 0.5, harvested by centrifugation, resuspended in sorbitol breaking buffer (0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl₂, 10 mM Tris [pH 7.5], 1× Boehringer Mannheim complete protease inhibitors), and lysed with glass beads, and extracts were cleared with a low-speed spin at 4°C. Protein concentration was measured in 1% sodium dodecyl sulfate (SDS) by the method of Lowry et al. (40). Protein extracts were solubilized in an equal volume of sample buffer (40 mM Tris-HCl [pH 6.8], 8 M urea, 15% SDS, 0.1 mM EDTA, 1% -mercaptoethanol, 0.01% bromophenol blue) and heated to 37°C for 20 min. Equal amounts of protein were subjected to electrophoresis on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with the antibody indicated below. For immunodetection of Yor1p, the C-terminal antibody was used for all Western blot and indirect immunofluorescence analyses and the N-terminal antibody was employed for all immunoprecipitation experiments. Immunoreactive material was detected with a donkey anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Amersham) and by chemiluminescence (Pierce).

Immunoprecipitation, pulse-labeling, and chase. Labeling and immunoprecipitations were performed essentially as described in reference 11 with the following modifications. Cells were grown overnight in complete synthetic medium to an OD₆₀₀ of 0.3 to 0.5. Cells lacking a temperature-sensitive allele were then harvested, resuspended in fresh prewarmed medium without methionine to an OD₆₀₀ of 2, and incubated for 10 min with gentle shaking. Cells were metabolically labeled by adding 15 µCi of Express protein labeling mix (New England Nuclear) per OD₆₀₀ unit of cells and shaking at 30°C for 10 min. A 100× chase solution containing 100 mM ammonium sulfate, 0.3% L-cysteine, and 0.4% L-methionine was added to the culture following labeling, and aliquots (5 × 10⁷ cells) were removed. The sec12-3 cells were grown at 23°C and shifted to 37°C for 10 min to impose the sec12-3 block or left at 23°C to permit continued Sec12-3p function. Following the temperature shift, cells were labeled at either the permissive or the restrictive temperature for 10 min and chased as described above. Time point aliquots were transferred to chilled tubes, and sodium azide was added to a final concentration of 10 mM. Cells were washed once with 10 mM sodium azide and resuspended in 200 µl of lysis buffer (15 mM Tris [pH 7.6], 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1× Boehringer Mannheim complete protease inhibitors). Glass beads were added, and cells were lysed by vigorously vortexing them three times for 1 min each time, with 1-min cooling intervals. SDS was then added to 1%, and two more 1-min vortexing rounds were performed. The resulting protein extracts were diluted with 5 volumes of immunoprecipitation (IP) buffer (15 mM Tris [pH 7.6], 150 mM NaCl, 1% Triton X-100, 2 mM sodium azide) and cleared by centrifugation. Polyclonal anti-Yor1p (1:100 dilution of the anti-N-terminal antibody) was added to the cleared lysate and incubated at 4°C in an end-over-end rotator for 1 h. Following this incubation, preswollen protein A-Sepharose beads (Sigma) were added and incubated end-over-end overnight at 4°C. Immune complexes bound to protein A-Sepharose beads were washed three times with IP buffer containing 0.1% SDS and two times with detergent-free wash buffer (10 mM Tris [pH 7.6], 50 mM NaCl, 2 mM sodium azide), resuspended in 40 µl of sample buffer, and heated to 37°C for 20 min. Twenty microliters of each sample was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (7.5% acrylamide), fixed in 7.5% acetic acid, soaked in 1 M sodium salicylate (pH 6.0) for 30 min, dried, and subjected to autoradiography or quantitated by phosphorimaging (Packard Cyclone).

Cell fractionation and sucrose gradient density centrifugation. Cellular membranes were resolved by sucrose gradient density centrifugation essentially as described in references 35 and 53. Cells (100 ml) were grown to an OD₆₀₀ of 0.3 to 0.5, sodium azide and potassium fluoride were added to 10 mM, and then the cells were briefly chilled on ice and harvested. Cells were then washed once in 10 mM sodium azide-10 mM potassium fluoride-5 mM Tris (pH 7.6) and resuspended in 0.5 ml of STE10 plus protease inhibitors (10% sucrose, 10 mM Tris [pH 7.6], 10 mM EDTA, 1× Boehringer Mannheim complete protease inhibitors). Glass beads were added, and the suspension was vortexed for 2 min. An additional 1 ml of STE10 plus protease inhibitors was then added and vortexed, and the supernatant was transferred to a new tube. This lysate was clarified by centrifugation for 3 min at 300 × g. Three hundred microliters of this cleared supernatant was then loaded onto a linear sucrose gradient prepared by sequentially layering STE solutions containing 10, 20, 35, or 60% sucrose to form a 5-ml step gradient. When sucrose gradients were centrifuged in the presence of Mg²⁺, we used the same protocol except that the sucrose solutions were prepared in TM buffer (10 mM Tris [pH 7.6], 1 mM MgCl₂). Each gradient was then tipped to a horizontal position and allowed to diffuse for 5 h. The gradients were then returned to an upright position, loaded with extract, and subjected to 14 h of centrifugation in a SW55.1 rotor (Beckman) at 50,000 rpm and 4°C. Fractions (380 µl) were collected from the top of the gradient, and proteins were precipitated with trichloroacetic acid. Protein samples were neutralized with the addition of 10 µl of 1 M Tris base and resuspended by heating them in 50 µl of sample buffer at 37°C for 30 min. The protein composition of each fraction was then analyzed by SDS-PAGE followed by Western blotting with appropriate antibodies.

Indirect immunofluorescence. Immunofluorescence experiments were performed essentially as described previously (51, 67). Cells (50 ml) were grown to an OD₆₀₀ of approximately 0.5 in either yeast extract-peptone-dextrose or selective medium, at which point 5.8 ml of 37% formaldehyde was added directly to the culture. The culture was then shaken at 30°C for an additional 30 min. Cells were then harvested by centrifugation, resuspended in paraformaldehyde fixative (51) lacking MgCl₂, and gently shaken overnight at room temperature. Fixed cells were washed four times in 1 ml of sorbitol buffer (1.2 M sorbitol, 50 mM sodium phosphate [pH 6.5]) with 1% -mercaptoethanol and resuspended in 1 ml of sorbitol buffer minus -mercaptoethanol. Spheroplasting was performed by adding 20 µl of a 1-mg/ml solution of oxylyticase (Enzogenetics) and shaking the suspension for 20 min at 30°C. Spheroplasted cells were then washed three times in sorbitol buffer and permeabilized by adding SDS to 0.05% and incubating the cells at 30°C for 5 min. Permeabilized cells were then washed three times with sorbitol buffer, adsorbed to polyethylenimine-coated slides, and exposed to antibodies. Yor1p was visualized by decoration with an affinity-purified anti-Yor1p (C-terminal) antibody and an anti-rabbit fluorescein isothiocyanate-conjugated goat antibody (Organon Teknika). Confocal microscopy was performed by standard techniques at the University of Iowa Electron Microscopy Center with a Bio-Rad confocal imaging system fitted to a conventional microscope with a 100× lens objective. Images were filtered by Kalman averaging and merged. Images were obtained and processed identically.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Wild-type Yor1p localizes to the plasma membrane by indirect immunofluorescence. We have previously described the isolation of the YOR1 gene on the basis of its important role in mediating oligomycin resistance (33). From the striking sequence similarity between Yor1p and other ABC transporter proteins of the MRP subfamily, we speculated that Yor1p might mediate oligomycin resistance through action as a multidrug transporter protein. To gain insight into the possible mechanism of Yor1p function, the subcellular location of the protein was determined. Indirect immunofluorescence experiments were carried out with affinity-purified antibodies that recognize the carboxy terminus of Yor1p (33). Immunofluorescence was first performed on strain SEY6210 (YOR1) and its isogenic yor1-1::hisG derivative DKY7. As can be seen in Fig. 1, the wild-type strain showed a highly punctate staining pattern around the periphery of the cell similar to that seen for other plasma membrane-targeted ABC transporter proteins (11, 49). No specific immunofluorescence was visible in the yor1-1::hisG mutant cells, confirming that the staining pattern in wild-type cells was specific for Yor1p. Overproduction of Yor1p from a high-copy-number plasmid carrying the wild-type gene led to a dramatic increase in the level of plasma membrane staining by the anti-Yor1p antibody. This result is consistent with the increased presence of Yor1p at the plasma membrane, which correlates with the increased oligomycin resistance seen in these cells (33). We interpret these data as providing evidence for the plasma membrane being the functional site for Yor1p activity.

View larger version (109K): [in this window] [in a new window]   FIG. 1.   Yor1p localization by indirect immunofluorescence. Isogenic strains with various levels of the YOR1 gene were prepared for indirect immunofluorescence essentially as described previously (67). Strains carried the yor1-1::hisG allele (left panels), had a single copy of YOR1 (middle panels), or were transformed with the wild-type YOR1 gene carried on a 2 µm plasmid (right panels). Cells were labeled with affinity-purified rabbit anti-Yor1p C-terminal antibody, followed by incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody.

View larger version (65K): [in this window] [in a new window]   FIG. 2.   Biochemical fractionation of Yor1p. Wild-type cells were lysed with glass beads and fractionated over a sucrose gradient. Aliquots of each sucrose gradient fraction were precipitated with trichloroacetic acid, resuspended in Laemmli buffer, and electrophoresed by SDS-PAGE. The proteins were then transferred to nitrocellulose, and the resulting blot was probed with the indicated antisera. The relative positions of the light (top) and heavy (bottom) fractions of the sucrose gradient are indicated on the figure. Antisera employed listed on the right-hand side are as follows: Yor1p is the affinity-purified rabbit anti-Yor1p antibody used in Fig. 1, Pma1p corresponds to the plasma membrane ATPase protein (55), Sec61p is an integral membrane subunit of the translocon in the ER (59), Vps10p (also called Pep1p) is the Golgi apparatus- or prevacuole-localized carboxypeptidase Y (CPY) receptor (43), and Vph1p is the 100-kDa subunit of the vacuolar ATPase (42).

Mutagenesis of the first nucleotide binding domain of Yor1p. ABC transporters are defined by their characteristic nucleotide binding domains. Like other nucleotide binding proteins, ABC transporters contain Walker A and Walker B motifs (64). However, the sequence similarity of ABC transporters extends over the entire nucleotide binding domain and is further defined by the presence of a short peptide sequence (LSGGQ) that lies between the Walker A and B elements (26). While the spacing between these three functional motifs is similar in all ABC transporter proteins, different families of these related proteins have been identified based on variations in the lengths of sequences separating the Walker A, Walker B, and LSGGQ motifs. One of these groups has been referred to as the MRP family since the sequence of this ABC transporter protein originally served to define this strictly conserved spacing (8). Sequence analysis of Yor1p indicated that this protein was also a member of the MRP family of ABC transporters, and an alignment showing the conserved spacing and sequences of several members of this group is shown in Fig. 3.

View larger version (61K): [in this window] [in a new window]   FIG. 3.   Alignment of NBD1 regions from MRP family members. An alignment of the NBD1 segments from each of the indicated ABC transporter proteins is shown. The numbers refer to the respective positions along the polypeptide chain, and residues that are identical in at least three of the five selected proteins are boxed. The conserved structural motifs present in all ABC transporters are indicated by the heavy lines, and the sites of mutations in Yor1p NBD1 are denoted by arrows.

Functions and expression of mutant Yor1p derivatives. Each mutant form of Yor1p was analyzed for its relative ability to complement the oligomycin-hypersensitive phenotype of a strain lacking a functional YOR1 structural gene. Mutant YOR1 genes were introduced on low-copy-number plasmids into a strain carrying the yor1-1::hisG allele and analyzed by spot test assay on plates containing a gradient of oligomycin (Fig. 4). The F670 YOR1 allele was also introduced on a high-copy-number plasmid to determine if residual function could be detected when the mutant protein was overproduced.

View larger version (89K): [in this window] [in a new window]   FIG. 4.   Oligomycin resistance phenotypes of Yor1p and mutant variants. DKY7 (yor1-1::hisG) cells were transformed with low-copy-number plasmids bearing the genes that express the indicated forms of Yor1p or with the low-copy-number vector (pRS316). Transformants were grown to an A600 of approximately 1, and 1,000 cells were placed on YPGE medium containing a gradient of oligomycin (indicated by the wedge of increasing height). The plate was incubated at 30°C and photographed.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Steady-state levels of mutant Yor1p derivatives. Whole-cell extracts were prepared from DKY7 (yor1-1::hisG) cells expressing the indicated forms of Yor1p from low-copy-number plasmids. Protein (75 µg) was electrophoresed by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-Yor1p (A) or anti-Vph1p (B) as a loading control.

Localization of mutant Yor1p derivatives. The Western blot data above confirmed that the observed defects seen in these mutant forms of Yor1p were not due to an effect on the levels of synthesis of these proteins. The localization of the mutant transporter proteins was next assessed by biochemical fractionation on sucrose gradients. The positions of the various forms of Yor1p and marker proteins of known subcellular distribution were determined by Western blotting (Fig. 6).

View larger version (43K): [in this window] [in a new window]   FIG. 6.   Subcellular fractionation of Yor1p NBD1 mutants. Cells lacking a chromosomal YOR1 locus (DKY7) were transformed with low-copy-number plasmids bearing the genes that express the indicated forms of Yor1p. Lysates were prepared and centrifuged through sucrose gradients as described in the legend to Fig. 2. Aliquots of each sucrose gradient were then subjected to Western blotting with the affinity-purified anti-Yor1p antibody (Yor1p) or the rabbit anti-Pma1p (Pma1p), rabbit anti-Vps10p (Vps10p), or rabbit anti-Sec61p (Sec61p) antiserum as listed on the right-hand side of each panel. The orientation of the sucrose gradient fractions is indicated as in Fig. 2.

The stability of F670 Yor1p is reduced relative to that of the wild-type protein. The reduced steady-state levels of F670 Yor1p suggested that this mutant derivative might be less stable than the corresponding wild-type protein. To directly test this possibility, pulse-chase analysis was carried out on strains expressing either the wild-type or F670 form of Yor1p. Cells were labeled with [³⁵S]methionine and then incubated in a large excess of unlabeled amino acids. Aliquots were withdrawn at various times, and Yor1p was recovered by immunoprecipitation. Immunoprecipitated material was resolved by SDS-PAGE, followed by autoradiography or phosphorimaging (Fig. 7).

View larger version (63K): [in this window] [in a new window]   FIG. 7.   Vacuolar proteases influence the turnover of both wild-type and F670 Yor1p. Isogenic yor1-1::hisG strains that either contained (top panels) or lacked (bottom panels) the PEP4 locus were transformed with low-copy-number plasmids bearing the gene that expresses the wild-type or F670 form of Yor1p. Selected transformants were then analyzed by pulse-chase analysis followed by immunoprecipitation with the anti-Yor1p antiserum or an anti-CPY polyclonal antibody (from R. Piper). Levels of immunoprecipitated proteins were quantitated by phosphorimaging. The time scale for the pulse-chase in the PEP4 background was in minutes, while that for the pulse-chase with the pep4 strain was in hours. The position of Yor1p and the positions of the various forms of CPY (58) are denoted by arrows.

View larger version (39K): [in this window] [in a new window]   FIG. 8.   Mg2+-dependent fractionation of F670 Yor1p in PEP4 or pep4 cells. Lysates were prepared from PEP4 (A) or pep4 (B) cells expressing either wild-type or F670 Yor1p. These lysates were subjected to sucrose gradient centrifugation in the presence or absence of Mg2+ as described previously (53). Aliquots of the gradient were then analyzed by Western blotting (see Materials and Methods) for the presence of either form of Yor1p and for Sec61p or Vph1p.

View larger version (38K): [in this window] [in a new window]   FIG. 9.   Effect of loss of Sec12p function on turnover of wild-type and F670 Yor1p. A strain containing the temperature-sensitive sec12-3 allele and lacking the YOR1 gene was constructed by one-step gene disruption of the YOR1 locus to produce EAE18. Low-copy-number plasmids bearing the gene that expresses either the wild-type or F670 form of Yor1p were then introduced into this gene background. Turnover of these two forms of Yor1p was then assessed by pulse-chase immunoprecipitation analysis at the permissive (23°C) and restrictive (37°C) temperatures. The numbers refer to times in minutes after chase addition.

Proteasome-mediated turnover of F670 Yor1p. The simplest explanation for the observed stabilization of F670 Yor1p by a pep4 mutation is a direct role for vacuolar proteases in the turnover of this protein. However, it is also possible that the pep4 defect acts indirectly to deplete a component required for normal proteasome-mediated turnover of proteins trapped in the ER. A candidate for such an indirect action is depletion of ubiquitin, as the vacuole is an important site for degradation of ubiquitinated proteins that arrive here after endocytosis from the plasma membrane (reviewed in reference 23). The absence of degradation has the potential to affect ubiquitin metabolism in a fashion that may depress degradation of F670 Yor1p.

View larger version (18K): [in this window] [in a new window]   FIG. 10.   F670 but not wild-type Yor1p is responsive to changes in ubiquitin metabolism. (A) A strain lacking the PEP4 gene was transformed with a 2 µm plasmid containing a CUP1-UBI1 gene fusion along with low-copy-number plasmids bearing the gene for either wild-type or F670 Yor1p. Levels of immunoprecipitable Yor1p were determined by pulse-chase analysis either in the absence (open symbols) or the presence (filled symbols) of copper sulfate to induce ubiquitin expression. The percentage of Yor1p remaining after chase was plotted as a function of time. The half-life (T1/2) of each immunoprecipitable Yor1p form is shown on the right-hand side in minutes. (B) An isogenic pair of yor1-1::hisG cells either lacking (ubc7) or containing (UBC7) an intact copy of chromosomal UBC7 was transformed with low-copy-number plasmids bearing the gene for wild-type or F670 Yor1p. Yor1p stability was analyzed by pulse-chase analysis as described above. The filled symbols indicate the presence of UBC7, while the open symbols correspond to a strain carrying a ubc7-1::HIS3 allele (32). The half-lives (in minutes) of the Yor1p forms are shown in the column on the right.

View larger version (11K): [in this window] [in a new window]   FIG. 11.   A functional proteasome is required for rapid degradation of F670 Yor1p. A yor1-1::hisG mutant cell lacking normal proteasome function (pre1-1 pre2-2) was transformed with low-copy-number plasmids bearing the gene for either the wild-type or F670 form of Yor1p. Low-copy-number plasmids carrying PRE1 and PRE2 (pPRE1/pPRE2) or the vector plasmids alone (pRS313/pRS315) were introduced into these transformants to examine the consequence of varying the activity of the proteasome on turnover of these two forms of Yor1p. Appropriate transformants were analyzed for stability of wild-type or F670 Yor1p by pulse-chase analysis as described above. The calculated half-lives (T1/2; in minutes) are on the right.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Polarized epithelial cells express both MRP and a closely related protein designated the multispecific organic anion transporter (c-MOAT) (48). In these polarized cells, MRP and c-MOAT are localized to the basolateral and apical membranes, respectively (reviewed in reference 34). Analyses of the complement of ABC transporters found in the S. cerevisiae genome have shown that a family of MRP-related transporters, including Yor1p and Ycf1p, is present in this organism (10, 44). This work has identified Yor1p as a plasma membrane-targeted transporter. Previous studies have defined Ycf1p as a component of the vacuolar membrane (37, 67). Thus, S. cerevisiae expresses a family of MRP-related ABC transporters and these transporters are destined for delivery to different membrane locations in the cell. These results illustrate the conserved physiology of the MRP family of proteins between animal cells and S. cerevisiae, supporting the notion that analysis of this fungal MRP family will provide valuable insight into the function of the cognate mammalian proteins.

One of the most intensively studied integral membrane proteins with respect to its intracellular trafficking is the human CFTR (52), associated with the disease CF. Most CFTR alleles associated with CF, including the most common CF-associated allele, F508, have the effect of causing CFTR to be trapped in the ER (62, 66). We probed NBD1 of Yor1p by mutagenesis to determine if this segment of the protein was similarly important in the biogenesis of this closely related yeast ABC transporter protein.

The F670 allele of Yor1p produced a transporter protein that exhibited trafficking behavior striking in its similarity to that of the F508 CFTR. These experiments suggest that F670 Yor1p is trapped in the ER in a fashion analogous to that of F508 CFTR (7). At least two explanations can be proposed to explain the consequences of the loss of this NBD1 phenylalanine codon: (i) important primary sequence information is deleted and (ii) spatial relationships are disturbed in the deletion mutants. The finding that the spacing of the functional motifs in Yor1p NBD1 is shorter by 1 amino acid than in the other members of the MRP family (33) suggested that the spacing within this region might be flexible. To investigate this possibility, we inserted an alanine residue at the gap in Yor1p NBD1 to convert the spacing of this protein to that seen for the other MRP members. This mutant exhibited trafficking behavior that was reminiscent of that of the wild-type CFTR. CFTR matures very slowly in the ER, with approximately 75% of the protein never reaching the plasma membrane (41). Analysis of the insA652 Yor1p derivative suggests that this mutant protein is much slower to leave the ER than wild-type Yor1p but that a small amount is capable of reaching the plasma membrane. This is seen both in the immunoreactive insA652 Yor1p being in the densest fractions of the sucrose gradient and in the weak but reproducible oligomycin resistance conferred by this mutant factor. In contrast, F670 Yor1p could not be detected in the dense sucrose gradient fractions and failed to complement the oligomycin hypersensitivity of a yor1 strain.

One of the most surprising features of F670 Yor1p was the stabilization of this protein that occurred upon loss of the PEP4 gene. Vacuolar proteases have not been observed to affect the stability of other integral membrane proteins that are degraded in the ER (14, 19, 38, 50). The rescue of this apparent stabilization by overexpression of ubiquitin has at least two possible explanations. First, it is possible that F670 Yor1p turnover is especially sensitive to levels of ubiquitin that may be slightly reduced in pep4 mutant strains. Elevation of ubiquitin levels may replenish whatever ubiquitin pool has been depleted. Second, vacuolar protease function may be required to produce an activity important in F670 Yor1p turnover at the ER. This possibility has precedent, as degradation of F508 CFTR has been found to involve several distinct proteolytic systems (29). Overproduction of ubiquitin might enhance proteasome function and allow accelerated turnover of F670 Yor1p by this proteolytic system, although under normal conditions, the proteasome would be one of several contributors to the degradation of F670 Yor1p. The recent demonstration that proteins can leave the yeast vacuole (6) is consistent with the notion that the vacuole may provide protein maturation as well as degradation function. Experiments are under way to further investigate the role of pep4 on F670 Yor1p turnover.

A final issue concerning the degradation of F670 Yor1p is the precise localization of this factor. While our data support the belief that this mutant protein is retained in the ER, we cannot eliminate the possibility that F670 Yor1p is found in intracellular locations in a Sec12p-independent fashion, where it is then degraded. Indirect immunofluorescence has failed to detect F670 Yor1p (data not shown), although this protein can easily be assayed by other immunological methods (Fig. 5 and 7). We are now constructing green fluorescent protein fusions to F670 Yor1p to directly visualize the subcellular distribution of this protein. F508 CFTR has recently been demonstrated to localize to unusual pericentriolar structures in cells unable to fully degrade this mutant protein (30). It will be important to determine if F670 Yor1p is also found in these aggresome structures.

Substitutions in the basic residue immediately following the LSGGQ motif in the MRP family members CFTR and Ycf1p have been found to have dramatic effects on the function of the resulting mutant proteins. A Ste6p-CFTR chimera containing CFTR NBD1, which failed to function in the presence of the F508 form of NBD1, regained activity when either Q or M replaced the arginine downstream of the NBD1 LSGGQ (61). A Ycf1p derivative containing a mutation analogous to F508 (Ycf1p F713), did not respond to Q or M replacement of the lysine following its NBD1 LSGGQ (67). Interestingly, an otherwise wild-type Ycf1p containing the K758Q or K758M lesion exhibited markedly increased function (67). These data from analyses of other MRP family members led us to expect that analogous replacements in Yor1p would elevate its biological function. However, both Yor1p K715Q and K715M are defective in function relative to that of the wild-type protein and K715Q Yor1p exhibits a modest defect in normal plasma membrane fractionation as evidenced by an elevated level of Yor1p immunoreactivity in the region of the sucrose gradient corresponding to the ER. Taken together, these data are consistent with the basic residue following the NBD1 LSGGQ motif being a key determinant in the folding and transport of the MRP family of ABC transporters. In certain instances (CFTR, Ycf1p), alteration of this residue can make the protein fold or be transported more rapidly, while in other instances (Yor1p), alterations of this position depress the ability to fold and/or transport.

Irrespective of the exact explanation behind the observed defects elicited by the NBD1 mutations that we have generated in Yor1p, alterations in this domain of Yor1p have dramatic effects on the function of the resulting mutant transporter protein. This sensitivity can be contrasted with the resistance of the a-factor transporter Ste6p to changes in function caused by alterations in the primary sequence of its NBD1 region (4). Two different single amino acid deletions in Ste6p NBD1 were found to have no detectable effect on the function of the resulting mutant protein. This comparison serves to illustrate the importance of the conserved spacing and high sequence conservation in the MRP family of proteins compared to that in other ABC transporter proteins of similar overall structure.

With Yor1p and CFTR, alterations in the NBD1 region often lead to a defect in transport from the ER. In the case of the F alleles of CFTR or Yor1p, the resulting trafficking defect also results in enhanced proteolysis of the mutant protein. Both of the F variants become substrates for ubiquitin-dependent proteolysis catalyzed by the proteasome. The finding of the similarity in molecular phenotype exhibited by F508 CFTR and F670 Yor1p provides the opportunity for genetic analysis of the mechanisms underlying the cell biology of the intracellular handling of this important class of ABC transporters.