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 end3Δ and doa4Δ 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.
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Acknowledgments
We thank Lucy Robinson for providing strains, helpful in-depth discussions, and critical reading of the manuscript and Xin Wang for providing pUN30-MAL61/HA-GFP. We also thank James V. Falvo and Erin O’Shea for providing us with the strain carrying the SNF7-RFP fusion gene and Aaron Mitchell for providing plasmid pWX307. Confocal imaging and fluorescence analysis and Western blot quantification were done in the Core Facilities for Cell and Molecular Biology of Queens College—CUNY. We greatly appreciate the assistance of Areti Tsiola for the same. This work was carried out in partial fulfillment of the requirements of the Ph.D. degree from the Graduate School of CUNY (N. G.) and was supported by a grant from the NIH (GM28216) to C. A. M.
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Gadura, N., Michels, C.A. Sequences in the N-terminal cytoplasmic domain of Saccharomyces cerevisiae maltose permease are required for vacuolar degradation but not glucose-induced internalization. Curr Genet 50, 101–114 (2006). https://doi.org/10.1007/s00294-006-0080-3
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DOI: https://doi.org/10.1007/s00294-006-0080-3