Characterization of the Plasmodium berghei regulatory AAA-ATPase subunit Rpt3 as an activator of Protein Phosphatase 1: direct and indirect evidence

Laine, C.; De Witte, C.; Martoriati, A.; Farce, A.; Metatla, I.; Guerrera, I. C.; Cailliau, K.; Khalife, J.; Pierrot, C.

doi:10.1101/2023.07.20.549862

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bioRxiv · 10.1101/2023.07.20.549862

Characterization of the Plasmodium berghei regulatory AAA-ATPase subunit Rpt3 as an activator of Protein Phosphatase 1: direct and indirect evidence

Laine, C.·De Witte, C.·Martoriati, A.·Farce, A.·Metatla, I.·Guerrera, I. C.·Cailliau, K.·Khalife, J.·Pierrot, C.

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Abstract

The 26S proteasome is the main proteolytic machine involved in protein degradation, thus contributing to homeostasis or stress response of eukaryotic cells. This macromolecular complex, consisting of a 20S core particle assembled with one or two 19S regulatory particles, is highly regulated by phosphorylation. Here we describe the Plasmodium berghei proteasome AAA-ATPase regulatory subunit Rpt3 and show that it binds to protein phosphatase 1, the major parasite phosphatase. In addition, PbRpt3 regulates the activity of the phosphatase both in vitro and in a heterologous model of Xenopus oocytes. Using mutagenesis approaches, we observed that the RVXF motifs of PbRpt3 are involved in this binding and activity. Further use of Xenopus oocyte model and mutagenesis based on the 3D model that we established revealed that the binding capacity of PbRpt3 to ATP may also contribute to its phosphatase-regulating activity. In the parasite, reverse genetic studies suggested an essential role for PbRpt3 since no viable knock-out line could be obtained. Additionally, immunoprecipitation assays followed by mass spectrometry analyses using transgenic PbRpt3-tagged parasites not only confirmed that PbRpt3 belongs to the 19S regulatory particle of the proteasome, but also revealed potential interaction with proteins already shown to play a role in the phospholipid membrane dynamics.

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Laine, C., De Witte, C., Martoriati, A., Farce, A., Metatla, I., Guerrera, I. C., Cailliau, K., Khalife, J., Pierrot, C.. 2023-07-20. Characterization of the Plasmodium berghei regulatory AAA-ATPase subunit Rpt3 as an activator of Protein Phosphatase 1: direct and indirect evidence. https://doi.org/10.1101/2023.07.20.549862

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