Small GTP-binding protein Arl8b interacts with RUFY1 and regulates cargo sorting to lysosomes

Abstract

Thesis abstract Title: Small GTP-binding protein Arl8b interacts with RUFY1 and regulates cargo sorting to lysosomes Abstract: Lysosomes are membrane-bound organelles that degrade macromolecules, such as proteins, lipids, carbohydrates, and nucleic acids, to recycle molecular building blocks for reuse by cells. Lysosomes move bidirectionally on microtubule tracks and fuse with late endosomes, phagosomes, and autophagosomes, following which; lysosomes are re-formed to maintain their steady state numbers and cellular function. In the Golgi complex, newly synthesized lysosomal hydrolases are mannose 6-phosphate (M6P)-tagged and transported to the late endosomes via cation independent mannose 6-phosphate receptor (CI-M6PR), where the hydrolases dissociate from the receptor, and receptor is retrieved from the endosomes for recycling back to the trans- Golgi network (TGN). Arl8b, a small Arf-like (Arl) GTP-binding (G) protein, is a key player in regulating lysosome distribution and function. Arl8b interacts with PLEKHM2/SKIP and RUFY3 to regulate lysosome bidirectional motility on microtubule tracks, and the HOPS (HOmotypic fusion and Protein Sorting) complex and PLEKHM1 to mediate lysosome fusion with late endosomes and autophagosomes, respectively. In this thesis, we report that Arl8b localizes on an early/recycling compartment of the endocytic pathway, where it binds RUN and FYVE domain-containing protein 1 (RUFY1) to regulate cargo sorting to lysosomes. RUFY1 depletion causes a delay in CI-M6PR retrieval from endosomes to the TGN, which leads to a defect in sorting of newly synthesised M6P-modified lysosomal hydrolases. Insufficient enzymes in lysosomes result in compromised lysosomal activity, leading to the accumulation of degradative cargo and the enlargement of lysosomes. Mass spectrometric- based identification of RUFY1 interaction partners revealed that RUFY1 interacts with dynein- dynactin complex to facilitate the retrograde trafficking of CI-M6PR towards the Golgi. Additionally, we have characterized a new Arl8b effector and obtained preliminary information on its role in regulating lysosome distribution and autophagy. Overall, the thesis highlights the crucial role of Arl8b and its interactions in regulating various aspects of lysosome function and endocytic transport pathways, providing valuable insights into the molecular mechanisms governing these cellular processes.