title: The role of Rab proteins in phosphatidylinositol 3,5-bisphosphate- mediated cellular traffic and signalling
Creators: name: Bercea, Cristiana
Publisher: name: University of Liverpool Repository
Year: 2025

description: Patients suffering from the peripheral neuropathy Charcot-Marie-Tooth type 4B (CMT4B) present mutations in three proteins which belong to the same enzymatic complex acting to dephosphorylate the phosphoinositide lipids phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Either of the catalytically inactive myotubularin-related proteins (MTMR) 5 and 13 forms a complex with the catalytically active MTMR2 to enhance its phosphatase activity toward PI3P and PI(3,5)P2; additionally, MTMR5 and 13 act as activators of small GTPases (involved in protein recruitment to intracellular membranes) through their N-terminal DENN domain. Previous studies isolated the small GTPase Rab35 as a strong interactor with Sbf, the Drosophila melanogaster homologue for MTMR13, but did not pursue this interaction. As it indicates a potential role for the mammalian Rab35 in MTMR13-mediated processes, and thus in CMT4B, I aimed to investigate the interaction of MTMR13 with Rab35 in mammalian cells in the context of CMT4B. We first looked at a CMT4 disease mutant of MTMR13 lacking the last 80 amino acids of the DENN domain (MTMR13Δ) and found that while Rab35 interacts with MTMR13, it does not with MTMR13Δ. We continued to study the interaction by truncating MTMR13; pull-downs with Rab35 show that the DENN domain or the linker between the DENN domain and the subsequent GRAM domain independently do not bind Rab35, indicating that they are necessary but not sufficient for the interaction. However, when we truncated the protein after the first 869 amino acids, thus isolating the DENN domain and the DENN-GRAM linker (a construct we refer to as 869*), this construct strongly interacts with Rab35. Pull-downs with constitutively active or dominant negative Rab35 show that 869* binds both forms, possibly indicating that 869* traps Rab35 in an intermediate stage. 869* localises to dynamic intracellular structures resembling circles, dots, and tubules, and recruits Rab35 to these specific locations. 869* frequently encircles (i.e. appears to coat) markers of lysosomes and occasionally colocalises with them (i.e. appears to be on the same membrane). Under starvation it colocalises or is contiguous with LC3-II-positive structures, and we observed it triggers a delay in the formation of LC3-II punctae in cell expressing 869*. In order to distinguish between endosomal and lysosomal compartments, we treated cells expressing 869* with dextran. After washout dextran-positive vesicles are coated in 869*. Dextran does not exit these compartments and there is a decrease in dextran intensity, indicating it is degraded. Therefore 869* possibly coats terminal stations. LAMP1-positive structures that are either coated by a circular 869* structure or co-localise with 869*-positive rods appear enlarged and are dynamic: a tube coming off an 869* structure surrounding LAMP1 or co-localising with LAMP1 appears to transport LAMP1. We also observe no homotypic fusion of LAMP1 structures when one of them colocalises with, or encircles, 869*, and less dynamic LAMP1-positive structures when coated with 869* than when colocalising with 869* tubules. Lastly, LAMP2 is redistributed to the cell periphery. We further studied the structure of 869*-positive organelles using correlative light and electron microscopy. We found that 869* structures are coated in a filamentous array resembling a helical polymer, similar to complexes containing BAR-domain proteins, such as the retromer. Taken together our data indicate that 869* is likely involved in autophagosome maturation and fusion with lysosomes, and thus may be an intermediate in the formation of autolysosomes.