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Rab12 Localizes to Shiga Toxin‐Induced Plasma Membrane Invaginations and Controls Toxin Transport
Author(s) -
Rydell Gustaf E.,
Renard HenriFrançois,
GarciaCastillo MariaDaniela,
Dingli Florent,
Loew Damarys,
Lamaze Christophe,
Römer Winfried,
Johannes Ludger
Publication year - 2014
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/tra.12173
Subject(s) - biology , toxin , shiga toxin , microbiology and biotechnology , transport protein , biochemistry , escherichia coli , gene
Several exogenous and endogenous cargo proteins are internalized independently of clathrin, including the bacterial Shiga toxin. The mechanisms underlying early steps of clathrin‐independent uptake remain largely unknown. In this study, we have designed a protocol to obtain gradient fractions containing Shiga toxin internalization intermediates. Using stable isotope labeling with amino acids in cell culture ( SILAC ) and quantitative mass spectrometry, Rab12 was found in association with these very early uptake carriers. The localization of the GTPase on Shiga toxin‐induced plasma membrane invaginations was shown by fluorescence microscopy in cells transfected with GFP ‐Rab12. Furthermore, using a quantitative biochemical assay, it was found that the amount of receptor‐binding B‐subunit of Shiga toxin reaching the trans ‐Golgi/ TGN membranes was decreased in Rab12‐depleted cells, and that cells were partially protected against intoxication by Shiga‐like toxin 1 under these conditions. These findings demonstrate the functional importance of Rab12 for retrograde toxin trafficking. Among several other intracellular transport pathways, only the steady‐state localizations of TGN46 and cation‐independent mannose‐6‐phosphate receptor were affected. These data thus strongly suggest that Rab12 functions in the retrograde transport route.