title: Dynamics and retention of misfolded proteins in native ER membranes.
abstract: When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40 degrees C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.
authors.affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, MD 20892, USA.
authors.forename: S
authors.initials: S
authors.lastname: Nehls
authors.forename: E L
authors.initials: EL
authors.lastname: Snapp
authors.forename: N B
authors.initials: NB
authors.lastname: Cole
authors.forename: K J
authors.initials: KJ
authors.lastname: Zaal
authors.forename: A K
authors.initials: AK
authors.lastname: Kenworthy
authors.forename: T H
authors.initials: TH
authors.lastname: Roberts
authors.forename: J
authors.initials: J
authors.lastname: Ellenberg
authors.forename: J F
authors.initials: JF
authors.lastname: Presley
authors.forename: E
authors.initials: E
authors.lastname: Siggia
authors.forename: J
authors.initials: J
authors.lastname: Lippincott-Schwartz
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D019556:COS Cells; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism*; D017930:Genes, Reporter; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014415:Tunicamycin / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; C028391:G protein, vesicular stomatitis virus; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D014415:Tunicamycin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D004229:Dithiothreitol