Requirement of the N-terminal extension for vacuolar trafficking and transport activity of yeast Ycf1p, an ATP-binding cassette transporter.
Ycf1p is the prototypical member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette (ABC) transporters. Ycf1p resides in the vacuolar membrane and mediates glutathione-dependent transport processes that result in resistance to cadmium and other xenobiotics. A feature common to many MRP proteins that distinguishes them from other ABC transporters is the presence of a hydrophobic N-terminal extension (NTE), whose function is not clearly established. The NTE contains a membrane spanning domain (MSD0) with five transmembrane spans and a cytosolic linker region (L0). The goal of this study was to determine the functional significance of the NTE of Ycf1p by examining the localization and functional properties of Ycf1p partial molecules, expressed either singly or together. We show that MSD0 plays a critical role in the vacuolar membrane trafficking of Ycf1p, whereas L0 is dispensable for localization. On the other hand, L0 is required for transport function, as determined by monitoring cadmium resistance. We also examine an unusual aspect of Ycf1p biology, namely, the posttranslational proteolytic processing that occurs within a lumenal loop of Ycf1p. Processing is shown to be Pep4p dependent and thus serves as a convenient marker for proper vacuolar localization. The processed fragments associate with each other, suggesting that these natural cleavage products contribute together to Ycf1p function.
Mason
Deborah L
DL
Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Michaelis
Susan
S
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002104:Cadmium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D005838:Genotype; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D008164:Luminescent Proteins; D029701:Saccharomyces cerevisiae Proteins; C089179:YCF1 protein, S cerevisiae; D002104:Cadmium; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D003513:Cycloheximide; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases