Overexpression of UBA5 in Cells Mimics the Phenotype of Cells Lacking UBA5

Sujata Kumari, Sayanika Banerjee, Manoj Kumar, Arata Hayashi, Balakrishnan Solaimuthu, Einav Cohen-Kfir, Yoav D. Shaul, Alexander Rouvinski, Reuven Wiener*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Ufmylation is a posttranslational modification in which the modifier UFM1 is attached to target proteins. This conjugation requires the concerted work of three enzymes named UBA5, UFC1, and UFL1. Initially, UBA5 activates UFM1 in a process that ends with UFM1 attached to UBA5’s active site Cys. Then, in a trans-thiolation reaction, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the final destination—a lysine residue on a target protein. Therefore, not surprisingly, deletion of one of these enzymes abrogates the conjugation process. However, how overexpression of these enzymes affects this process is not yet clear. Here we found, unexpectedly, that overexpression of UBA5, but not UFC1, damages the ability of cells to migrate, in a similar way to cells lacking UBA5 or UFC1. At the mechanistic level, we found that overexpression of UBA5 reverses the trans-thiolation reaction, thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, as seen in cells lacking UBA5, reduces the level of charged UFC1 and therefore harms the conjugation process. In contrast, co-expression of UBA5 with UFM1 abolishes this effect, suggesting that the reverse transfer of UFM1 from UFC1 to UBA5 depends on the level of free UFM1. Overall, our results propose that the cellular expression level of the UFM1 conjugation enzymes has to be tightly regulated to ensure the proper directionality of UFM1 transfer.

Original languageAmerican English
Article number7445
JournalInternational Journal of Molecular Sciences
Volume23
Issue number13
DOIs
StatePublished - 4 Jul 2022

Bibliographical note

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • E1-activating enzymes
  • E2-conjugating enzymes
  • UBA5
  • UFC1
  • UFM1
  • ubiquitin-like protein
  • ufmylation

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