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

Arpita Sen, Wen-Chieh Hsieh, Claudia B. Hanna, Chuan-Chih Hsu, McKeith Pearson II, W. Andy Tao, R. Claudio Aguilar
doi: https://doi.org/10.1101/2020.02.07.939082
Arpita Sen
1Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
2Aromyx Inc., 319 N. Bernardo Avenue, Mountain View, CA 94043, USA
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Wen-Chieh Hsieh
1Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
3Section on Cellular Communication, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
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Claudia B. Hanna
1Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
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Chuan-Chih Hsu
4Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
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McKeith Pearson II
1Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
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W. Andy Tao
4Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA
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R. Claudio Aguilar
1Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
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  • For correspondence: Claudio@purdue.edu
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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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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 08, 2020.
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Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae
Arpita Sen, Wen-Chieh Hsieh, Claudia B. Hanna, Chuan-Chih Hsu, McKeith Pearson II, W. Andy Tao, R. Claudio Aguilar
bioRxiv 2020.02.07.939082; doi: https://doi.org/10.1101/2020.02.07.939082
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Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae
Arpita Sen, Wen-Chieh Hsieh, Claudia B. Hanna, Chuan-Chih Hsu, McKeith Pearson II, W. Andy Tao, R. Claudio Aguilar
bioRxiv 2020.02.07.939082; doi: https://doi.org/10.1101/2020.02.07.939082

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