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Investigating endocytic pathways to the endoplasmic reticulum and to the cytosol using SNAP-trap


Geiger, Roger; Luisoni, Stefania; Johnsson, Kai; Greber, Urs F; Helenius, Ari (2013). Investigating endocytic pathways to the endoplasmic reticulum and to the cytosol using SNAP-trap. Traffic, 14(1):36-46.

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.

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.

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5 citations in Web of Science®
6 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2013
Deposited On:25 Oct 2012 08:24
Last Modified:05 Apr 2016 16:01
Publisher:Wiley-Blackwell
ISSN:1398-9219
Publisher DOI:10.1111/tra.12018
PubMed ID:23046100

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