title: Investigating endocytic pathways to the endoplasmic reticulum and to the cytosol using SNAP-trap.
abstract: Cholera toxin enters cells via an unusual pathway that involves trafficking through endosomes to the endoplasmic reticulum (ER). Whether the toxin induces its own pathway or travels along a physiological retrograde route is not known. To study its trafficking, we labeled cholera toxin B (CTB) or endogenous plasma membrane proteins with a small chemical compound, benzylguanine, which covalently reacts with the protein SNAP-tag. Using ER-targeted SNAP-tag as reporter, we found that transport of CTB to the ER depends on dynamin-2 and syntaxin 5. Plasma membrane proteins and a fluid-phase marker added to the medium were also transported to the ER. This flux was not affected by exposing cells to CTB but was inhibited by depleting syntaxin 5 and increased by depleting dynamin-2. As a control for confined intracellular localization of ER-targeted SNAP-tag we used adenovirus-5, which traffics to endosomes and then escapes into the cytosol. The virus did not react with ER-targeted SNAP but with cytosolic SNAP. Together, our results establish a new method (SNAP-trap) to study trafficking of different cargo to the ER and the cytosol and provide evidence for the existence of a constitutive pathway from the cell surface to the ER.
authors.affiliation: Institute of Biochemistry, ETH Zurich, Schafmattstrasse 18, Zurich, CH-8093, Switzerland. roger.geiger@irb.usi.ch
authors.forename: Roger
authors.initials: R
authors.lastname: Geiger
authors.forename: Stefania
authors.initials: S
authors.lastname: Luisoni
authors.forename: Kai
authors.initials: K
authors.lastname: Johnsson
authors.forename: Urs F
authors.initials: UF
authors.lastname: Greber
authors.forename: Ari
authors.initials: A
authors.lastname: Helenius
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D034285:Dynamin II