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bioRxiv · 10.1101/2020.02.07.939082

Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae

Sen, A.·Hsieh, W.-C.·Hanna, C. B.·Hsu, C.-C.·Pearson, M.·Tao, A.·Aguilar, R. C.
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Abstract

It is well-known that in addition to its classical role in protein turnover, ubiquitination is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin (Ub) units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Here we provide an answer to this question as we discovered a mechanism based on the coincidence detection of lysine ubiquitination and Ser/Thr phosphorylation for the endocytic adaptor epsin to mediate the internalization of the yeast Na+ pump Ena1. Internalization of Ena1-GFP was abolished in double epsin knock-out in S. cerevisiae and was rescued by re-introducing either one of the 2 yeast epsins, Ent1 or Ent2 in an UIM (Ub Interacting Motif)-dependent manner. Further, our results indicate that ubiquitination of its C-terminal Lys1090 is needed for internalization of Ena1 and requires the arrestin-related-trafficking adaptor, Art3. We determined that in addition to ubiquitination of K1090, the presence of a Ser/Thr-rich patch (S1076TST1079) within Ena1 was also essential for its internalization. Our results suggest that this ST motif is targeted for phosphorylation by casein kinases. Nevertheless, phosphorylation of this S/T patch was not required for ubiquitination. Instead, ubiquitination of K1090 and phosphorylation of the ST motif were independently needed for epsin-mediated internalization of Ena1. We propose a model in which a dual detection mechanism is used by Ub-binding elements of the sorting machinery to differentiate among multiple Ub-cargoes.

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Sen, A., Hsieh, W.-C., Hanna, C. B., Hsu, C.-C., Pearson, M., Tao, A., Aguilar, R. C.. 2020-02-08. Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae. https://doi.org/10.1101/2020.02.07.939082

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