Characterization of the regulatory AAA-ATPase subunit Rpt3 in Plasmodium berghei as an activator of Protein Phosphatase 1

ABSTRACT: The 26S proteasome is the main proteolytic machine involved in protein degradation, 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 phos-phorylation. Here we described the Plasmodium berghei (Pb) proteasome AAA-ATPase regulatory subunit Rpt3 and showed that it binds to protein phosphatase 1 catalytic subunit (PP1c), one of the major and essential parasite phosphatases. In addition, PbRpt3 protein was able to enhance the activity of PP1c both in vitro and in a heterologous model of Xenopus oocytes. Using mutagen-esis approaches, we observed that the RVXF motifs of PbRpt3 are involved in its binding and reg-ulatory function. Further use of Xenopus oocytes in combination with PbRpt3 protein mutated for amino acids predicted to be involved in ATP binding, suggested that the binding capacity of PbRpt3 to ATP may also contribute to its phosphatase-regulatory activity. In Pb, reverse genetic studies suggested an essential role for PbRpt3 since no viable knock-out line could be obtained. Finally, immunoprecipitation assays followed by mass spectrometry analyses using transgenic PbRpt3-tagged parasites confirmed that PbRpt3 belongs to the 19S regulatory particle of the pro-teasome and revealed potential interactions with proteins previously shown to be involved in membrane phospholipid binding.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Chiara guerrera  

LAB HEAD: Ida Chiara Guerrera

PROVIDER: PXD044616 | Pride | 2025-12-03

REPOSITORIES: Pride