title: Two separate signals act independently to localize a yeast late Golgi membrane protein through a combination of retrieval and retention.
abstract: The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins. These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN. To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A [DPAP A] fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase [ALP]), which localizes to the yeast TGN. Insertion of the aromatic residue-based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN. We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN. We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN. We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN. We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Bryant
authors.forename: T H
authors.initials: TH
authors.lastname: Stevens
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D014617:Vacuoles / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase; D007703:Peptidyl-Dipeptidase A