title: Identification of a novel saturable endoplasmic reticulum localization mechanism mediated by the C-terminus of a Dictyostelium protein disulfide isomerase.
abstract: Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.
authors.affiliation: Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, D-69120 Heidelberg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Monnat
authors.forename: E M
authors.initials: EM
authors.lastname: Neuhaus
authors.forename: M S
authors.initials: MS
authors.lastname: Pop
authors.forename: D M
authors.initials: DM
authors.lastname: Ferrari
authors.forename: B
authors.initials: B
authors.lastname: Kramer
authors.forename: T
authors.initials: T
authors.lastname: Soldati
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D004023:Dictyostelium / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000502:physiology* / Q000648:ultrastructure; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D008164:Luminescent Proteins; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; C077480:histidyl-aspartyl-glutamyl-leucine; D049452:Green Fluorescent Proteins; D003513:Cycloheximide; D019704:Protein Disulfide-Isomerases