title: Retrieval from the ER-golgi intermediate compartment is key to the targeting of c-terminally anchored ER-resident proteins.
abstract: Endoplasmic reticulum (ER) resident proteins may be maintained in the ER by retention, where the leak into post-ER compartments is absent or slow, or retrieval, where a significant leak is countered by retrieval from post-ER compartments. Here the targeting of the C-terminally anchored protein ER-resident protein, cytochrome b5a (cytb5a), considered to be maintained in the ER mainly by the process of retention, is compared with that of sarcolipin (SLN) and phospholamban (PLB); also C-terminally anchored ER-residents. Laser confocal microscopy, and cell fractionation of green fluorescent protein-tagged constructs expressed in COS 7 cells indicate that while calnexin appears to be retained in the ER with no evidence of leak into the ER-Golgi intermediate compartment (ERGIC), significant amounts of cytb5a, SLN, and PLB are detectable in the ERGIC, indicating that there is considerable leak from the ER. This is supported by an in vitro budding assay that shows that while small amounts of calnexin appear in the transport vesicles budding off from the ER, significant amounts of cytb5a and SLN are found in such vesicles. These data support the hypothesis that retrieval plays a major role in ensuring that C-terminally anchored proteins are maintained in the ER.
authors.affiliation: School of Biological Sciences, Building 85, University of Southampton, Southampton SO17 1BJ, UK.
authors.forename: John
authors.initials: J
authors.lastname: Butler
authors.forename: Helen R
authors.initials: HR
authors.lastname: Watson
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Lee
authors.forename: Hans-Jurgen
authors.initials: HJ
authors.lastname: Schuppe
authors.forename: J Malcolm
authors.initials: JM
authors.lastname: East
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin / Q000378:metabolism; D002451:Cell Compartmentation; D002522:Chlorocebus aethiops; D015786:Cytochromes b5 / Q000378:metabolism; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D037281:Calnexin; D015786:Cytochromes b5; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D011506:Proteins; D000099211:Phospholamban