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FERARI: A multi-protein tethering platform involved in endocytic recycling

Rashid, Harun-Or. FERARI: A multi-protein tethering platform involved in endocytic recycling. 2021, Doctoral Thesis, University of Basel, Faculty of Science.

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Abstract

Eukaryotic cells have developed intracellular membrane-bound organelles through evolution. These organelles maintain an extended communication system using shuttling vesicles or contact sites. Vesicles bud from the donor organelles or the plasma membrane and transport proteins, lipids, and nutrients across the cytosol to the acceptor membrane. This endomembrane trafficking is a thoroughly regulated process to guarantee correct cargo transport to the right destination organelle or the plasma membrane.
The sorting endosome is a hub for diverse trafficking pathways, including recycling towards plasma membrane, secretion, or protein degradation into the lysosome. Multiple fusion and fission events regulate protein and lipid transport along the eukaryotic endomembrane system. Multi-subunit tethering complexes contribute to the fusion of two opposing membranes with the help of Rab GTPases and SNARE proteins. Co-ordinated function of Rab GTPases, tethers, and SNAREs are required for membrane tethering and fusion. Studies in yeast have identified multiple tethering factors in regulation of membrane tethering and fusion. However, due to the diversification of the cargo and duplication of genes involved in cargo trafficking, membrane tethering is much more complex in higher eukaryotes, especially in the mammalian system. Furthermore, how cargo recycling is regulated from the sorting endosomes is largely obscure, and the involvement of any tethering complex in this process needs to be thoroughly investigated. The presented work is aimed to provide a better understanding of cargo recycling from sorting endosomes in connection with a multi-subunit tethering platform.
In parallel a work conducted in the with C. elegans, I have characterized a conserved six member-tethering platform, Factors for Endosome Recycling and Rab Interactions (FERARI) in the mammalian system. We found that, in human cell lines, the FERARI member VIPAS39 interacts with five other subunits: VPS45, Rabenosyn-5, EHD1, Rab11-FIP5 and ANK3. FERARI, as a platform, regulates cargo recycling from sorting endosomes via Rab11-positive recycling vesicles. Transferrin recycling is partially impaired when FERARI is depleted. Rab11-FIP5 and Rabenosyn-5 are two Rab interacting subunits of FERARI that bind Rab11 and Rab5, respectively. FERARI tethers Rab11 containing recycling vesicles to Rab5 positive endosomes, which is then followed by membrane fusion through SNARE proteins regulated by the FERARI subunit Sec1/Munc18 protein VPS45.
Surprisingly, unlike conventional tethering complexes, FERARI contains the membrane fission protein EHD1. Thus, FERARI probably couples fusion and fission activity of recycling vesicles on sorting endosomes through the SM protein VPS45 and membrane pinchase activity of EHD1 respectively. Interestingly, we also found that FERARI is associated with the BAR domain protein SNX1, which is involved in membrane tubulation. ANK3, an actin regulator associated with FERARI, might stabilize SNX1 positive compartments.
In the second part of this thesis, we focused on cargo uptake process by the recycling vesicles from sorting endosomes. Most notably, in HeLa cells, endogenously tagged GFP-Rab11 vesicles transiently associates with SNX1 positive structure and dissociates, suggesting a combination of kiss-and-run process. Furthermore, we have found that the cargo adaptor protein SNX6, but not SNX5, interacts with Rabenosyn-5 and VIPAS39. Combined knockout of snx5 and snx6 resulted in a FERARI like phenotype, enlarged Rab11 structure. While the association of SNX1 with Rab11 is reduced Rab5 colocalization was increased in snx5+6 KO cells.
In the third part of this thesis, I discuss the current study of purification of FERARI. To purify entire FERARI complex, we over-expressed GFP-Rabenosyn-5 in HEK-293 cells and pulled down other subunits heterologously expressed in yeast.
Finally, to gain insight into the biological significance of FERARI, we analyzed cilia development in retinal epithelial cells by knocking out FERARI subunits. We demonstrated that cilia size is shorter in FERARI depleted condition. Normally, Rab11 mediated polarized cargo trafficking is crucial for cilia development. However, in FERARI KO cell Rab11 is not efficiently recruited to the ciliary base. Overall, this work represents the first characterization of FERARI in mammalian cells.
Advisors:Spang, Anne
Committee Members:Spiess, Martin and Gruenberg, Jean
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spang)
UniBasel Contributors:Spang, Anne and Spiess, Martin
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14245
Thesis status:Complete
Number of Pages:147
Language:English
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss142458
edoc DOI:
Last Modified:10 Sep 2021 04:30
Deposited On:09 Sep 2021 07:35

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