Rab12 localizes to Shiga toxin-induced plasma membrane invaginations and controls toxin transport.
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.
Institut Curie, Centre de Recherche, Endocytic Trafficking and Therapeutic Delivery Group, 26 rue d'Ulm, 75248, Paris Cedex 05, France; CNRS UMR3666, 75005, Paris, France; U1143 INSERM, 75005, Paris, France.
Gustaf E
GE
Rydell
Henri-François
HF
Renard
Maria-Daniela
MD
Garcia-Castillo
Florent
F
Dingli
Damarys
D
Loew
Christophe
C
Lamaze
Winfried
W
Römer
Ludger
L
Johannes
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D022621:Shiga Toxin / Q000378:metabolism / Q000494:pharmacology*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D022621:Shiga Toxin; C000595432:Rab12 protein, human; D020691:rab GTP-Binding Proteins
RabGTPase; STxB; Shiga toxin B-subunit; clathrin-independent endocytosis; intracellular trafficking; membrane tubules; retrograde transport