BioID Reveals Novel Proteins of the Parasitophorous Vacuole Membrane.

Schnider, Cilly Bernardette; Bausch-Fluck, Damaris; Brühlmann, Francis; Heussler, Volker; Burda, Paul-Christian (2018). BioID Reveals Novel Proteins of the Parasitophorous Vacuole Membrane. mSphere, 3(1), e00522-17. American Society for Microbiology 10.1128/mSphere.00522-17

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During their development within the vertebrate host, parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells; however, only a limited number of proteins of this membrane have been identified so far. This is partially because systematic proteomic analysis of the protein content of the PVM has been difficult in the past, due to difficulties encountered in attempts to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to -cultivated blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our report expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in . Intracellular pathogens are often surrounded by a host-cell derived membrane. This membrane is modified by the pathogens to their own needs and is crucial for their intracellular lifestyle. In parasites, this membrane is referred to as the PVM and only a limited number of its proteins are known so far. Here, we applied in rodent parasites a method called BioID, which is based on biotinylation of proximal and interacting proteins by the promiscuous biotin ligase BirA*, and demonstrated its usefulness in identification of novel PVM proteins.

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