TY - JOUR
T1 - Rab12 Localizes to Shiga Toxin-Induced Plasma Membrane Invaginations and Controls Toxin Transport
AU - Rydell, Gustaf E.
AU - Renard, Henri François
AU - Garcia-Castillo, Maria Daniela
AU - Dingli, Florent
AU - Loew, Damarys
AU - Lamaze, Christophe
AU - Römer, Winfried
AU - Johannes, Ludger
PY - 2014/7
Y1 - 2014/7
N2 - 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. SILAC labeling and quantitative mass spectrometry were used to identify Rab12 on Shiga toxin-induced endocytic plasma membrane invaginations. Localization of GFP-Rab12 on these structures was shown by fluorescence microscopy. Shiga toxin trafficking to trans-Golgi/TGN membranes and toxin-mediated inhibition of protein biosynthesis were inhibited in Rab12-depleted cells, suggesting that Rab12 functions in the retrograde transport route.
AB - 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. SILAC labeling and quantitative mass spectrometry were used to identify Rab12 on Shiga toxin-induced endocytic plasma membrane invaginations. Localization of GFP-Rab12 on these structures was shown by fluorescence microscopy. Shiga toxin trafficking to trans-Golgi/TGN membranes and toxin-mediated inhibition of protein biosynthesis were inhibited in Rab12-depleted cells, suggesting that Rab12 functions in the retrograde transport route.
KW - Clathrin-independent endocytosis
KW - Intracellular trafficking
KW - Membrane tubules
KW - RabGTPase
KW - Retrograde transport
KW - Shiga toxin B-subunit
KW - STxB
UR - http://www.scopus.com/inward/record.url?scp=84902672939&partnerID=8YFLogxK
U2 - 10.1111/tra.12173
DO - 10.1111/tra.12173
M3 - Article
C2 - 24703428
AN - SCOPUS:84902672939
SN - 1398-9219
VL - 15
SP - 772
EP - 787
JO - Traffic
JF - Traffic
IS - 7
ER -