Phosphatidylinositol phosphates and their effector proteins in phagosome-lysosome tethering and fusion

• Applicant: Professor Dr. Albert Haas
• Subject Area: Biochemistry
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 414783339

Phagocytosis is a specialized form of endocytosis during which particles are ingested and delivered to phagolysosomes for degradation. Phagosomes develop into phagolysosomes as they sequentially fuse with early endosomes, late endosomes and lysosomes. To this end, phagosomes and endosomes/lysosomes need to move toward each other and to bind cognate sets of fusion factors. Small GTPases of the Rab- and Arf-families guide phagosome transport and fusion by anchoring motor proteins and fusion factors to phagosome membranes. Fusion between phagosomes and late endosomes or lysosomes depends on HOPS, a multi-subunit tethering complex which bridges compartment-specific Rab- and/or Arf-GTPases before fusion. Many proteins bind to HOPS and may link it to membranes. However, it is not clear which HOPS interactors are bridged during which fusion step of phagosome maturation. The proposed study will analyze the various fusion steps along phagosome maturation for a participation of HOPS and interacting proteins (i.e., Rab2a, Rab7a, Arl8, BORC, and SKIP) to determine which proteins cooperate with HOPS at which stage of the pathway. Additionally, the project will investigate how phosphatidylinositol phosphate lipids (PIPs) and the PIP-binding HOPS interactor SKIP contribute to assembly and membrane binding of HOPS. In particular, a biochemical assay that reconstitutes fusion of purified phagosomes and endosomes or lysosomes will be employed to determine the contributions of HOPS and its various interactors to fusion. This assay not only allows to identify fusion factors but also to assign the action of fusion factors to a specific sub-reaction of fusion, such as tethering. The project will further combine reconstituted stage-specific fusion reactions with co-immunoprecipitation and mass spectrometry to identify which proteins bind to HOPS during which fusion step of phagosome maturation in an unbiased fashion. Centering on phagocytosis, a central part of innate immunity, this study will not only help to understand the biochemistry of membrane fusion but will also provide target information for drug development, in particular against intracellular pathogens.

• DFG Programme: Research Grants