Abstract #62

Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination, ERAD and proteasome activity. Manfred Schmitt, Susanne Heiligenstein, Tanja Sendzik, Natalia Jimenez-Becker, Frank Breinig. FR 8.3 Mikrobiologie Geb A l.5, Angewandte Molekularbiologie, Saarbrucken, Germany.
   K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the ER requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p and proper maintenance of Ca²⁺ homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical dislocation via ERAD. We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where B is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity. [supported by grant Schm 541/11-2 from the Deutsche Forschungsgemeinschaft].

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