The E3 ubiquitin ligases Hrd1 and gp78 bind to and promote cholera toxin retro-translocation.
To cause disease, cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) lumen where the catalytic CTA1 subunit retro-translocates to the cytosol to induce pathological water secretion. Two retro-translocon components are the Derlins and ER-associated multi-spanning E3 ubiquitin ligases including Hrd1 and gp78. We demonstrated previously that Derlin-1 facilitates CTA1 retro-translocation. However, as CTA1 is neither ubiquitinated on lysines nor at its N-terminus, the role of E3 ligases in toxin retro-translocation is unclear. Here, we show that expression of mutant Hrd1 and gp78 and a mutant E2-conjugating enzyme dedicated to retro-translocation (Ube2g2) decrease CTA1 retro-translocation. Hrd1 knockdown also attenuated toxin retro-translocation. Binding studies demonstrate that Hrd1 and gp78 interact with CT and protein disulfide isomerase, an ER chaperone that unfolds CTA1 to initiate translocation. Moreover, we find that the toxin's association with Hrd1 and gp78 is blocked by dominant-negative Derlin-1, suggesting that CT is targeted initially to Derlin-1 and then transferred to Hrd1 and gp78. These data demonstrate a role of the E3 ubiquitin ligases in CTA1 retro-translocation, implicate a sequence of events experienced by the toxin on the ER membrane, and raise the possibility that ubiquitination is involved in the transport process.
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Kaleena M
KM
Bernardi
Jeffrey M
JM
Williams
Marjolein
M
Kikkert
Sjaak
S
van Voorden
Emmanuel J
EJ
Wiertz
Yihong
Y
Ye
Billy
B
Tsai
D001426:Bacterial Proteins / Q000378:metabolism; D055162:Biocatalysis; D002772:Cholera Toxin / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D001426:Bacterial Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D002772:Cholera Toxin; C471617:UBE2G2 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases; D019704:Protein Disulfide-Isomerases