title: Sequences in the N-terminal cytoplasmic domain of Saccharomyces cerevisiae maltose permease are required for vacuolar degradation but not glucose-induced internalization.
abstract: In Saccharomyces cerevisiae, glucose addition to maltose fermenting cells causes a rapid loss of maltose transport activity and ubiquitin-mediated vacuolar proteolysis of maltose permease. GFP-tagged Mal61 maltose permease was used to explore the role of the N-terminal cytoplasmic domain in glucose-induced inactivation. In maltose-grown cells, Mal61/HA-GFP localizes to the cell surface and, surprisingly, to the vacuole. Studies of end3Delta and doa4Delta mutants indicate that a slow constitutive internalization of Mal61/HA-GFP is required for its vacuolar localization. Site-specific mutagenesis of multiple serine/threonine residues in a putative PEST sequence of the N-terminal cytoplasmic domain of maltose permease blocks glucose-induced Mal61p degradation but does not affect the rapid loss of maltose transport activity associated with glucose-induced internalization. The internalized multiple Ser/Thr mutant protein co-localizes with Snf7p in a putative late endosome or E-compartment. Further, alteration of a putative dileucine [D/EExxxLL/I] motif at residues 64-70 causes a significant defect in maltose transport activity and mislocalization to an E-compartment but appears to have little impact on glucose-induced internalization. We conclude that the N-terminal cytoplasmic domain of maltose permease is not the target of the signaling pathways leading to glucose-induced internalization of Mal61 permease but is required for its subsequent delivery to the vacuole for degradation.
authors.affiliation: Biology Department, Queens College and the Graduate School of the City University of New York, Flushing, 11367, USA.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Gadura
authors.forename: Corinne A
authors.initials: CA
authors.lastname: Michels
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C021534:maltose permease; D005947:Glucose