Identification of a novel saturable endoplasmic reticulum localization mechanism mediated by the C-terminus of a Dictyostelium protein disulfide isomerase

Localization of soluble endoplasmic reticulum (ER) resident proteins is lik ely achieved by the complementary action of retrieval and retention mechani sms. Whereas the machinery involving the H/KDEL and related retrieval signa ls in targeting escapees back to the ER is well characterized, other mechan isms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal norm ally found at the C terminus of ER residents in Dictyostelium discoideum. H ere we demonstrate that its 57 residue C-terminal domain is necessary for i ntracellular retention of Dd-PDI and sufficient to localize a green fluores cent protein (GFP) chimera to the ER, especially to the nuclear envelope. D d-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. Th e overexpression of GFP-PDI57 leads to disruption of endogenous PDI complex es and induces the secretion of PDI, whereas overexpression of a GFP-HDEL c himera induces the secretion of endogenous calreticulin, revealing the pres ence of two independent and saturable mechanisms. Finally, low-level expres sion of Dd-PDI but not of PDI truncated of its 57 C-terminal residues compl ements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating fu nctional disulfide isomerase activity and ER localization. Altogether, thes e results indicate that the PDI57 peptide contains ER localization determin ants recognized by a conserved machinery present in D. discoideum and Sacch aromyces cerevisiae.