Search bioRxiv⌕ Search

bioRxiv · 10.1101/2024.05.13.593900

Rab7 deficiency induces lysosome formation from recycling endosomes leading to an increased degradation of cell surface proteins

Abstract

Cell surface receptors such as integrins are repeatedly internalized from and recycled back to the plasma membrane before routed to lysosomes for degradation. In search for modulators of {beta}1 integrin surface stability, we identified the Rab7 small GTPase, believed to be required for lysosome biogenesis, as integrin stabilizer. We show that Rab7 deficiency produces late endosomes and lysosomes with acidic pH, lysosome-specific proteins and membrane architectures that are functional in protein degradation and organelle fusion. Furthermore, Rab7-deficient lysosomes form from Rab4- and transferrin receptor-positive recycling endosomes, resulting in the degradation of proteins designated for recycling. Finally, we also found that overexpression of Rab4 can direct lysosome formation from recycling endosomes in absence as well as presence of Rab7, however, the latter to a much lesser extent. Our findings reveal a lysosome biogenesis and lysosomal protein degradation pathway that becomes dominant in absence of Rab7 or when Rab4 is highly abundant.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Wang, G. M., Xu, P., Yu, K., Guo, S. S., Faessler, R.. 2024-05-15. Rab7 deficiency induces lysosome formation from recycling endosomes leading to an increased degradation of cell surface proteins. https://doi.org/10.1101/2024.05.13.593900

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

Contact-phagy remodels ER-mitochondria contact sites

Membrane contact sites between the endoplasmic reticulum (ER) and other organelles coordinate cellular organization, metabolism and stress responses, yet how these interfaces are selectively remodeled and removed remains poorly understood. Here we identify a selective autophagy pathway that targets defined ER-mitochondria contact-site (ERMCS) subdomains for turnover, a process we term Contact-phagy. The ER-resident chaperone SIGMAR1 forms a functional complex with the ER-phagy receptor CCPG1, and SIGMAR1-CCPG1 nanoclusters define ERMCS-associated sites of autophagy initiation. Contact-phagy is selectively engaged by metabolic and mitochondrial stress and promotes turnover of defined contact-site components without broadly affecting general ER-phagy, mitophagy or bulk autophagy. At these sites, SIGMAR1-CCPG1 function is coupled to mitochondrial fission: MFF promotes DRP1 recruitment to ERMCS, whereas loss of CCPG1 or SIGMAR1 causes abnormal persistence and clustering of DRP1 and MFF, accompanied by altered mitochondrial remodeling. Loss of CCPG1 further disrupts ER-mitochondria organization and mitochondrial function in pancreatic tissue, while pancreatic cancer cells depend on CCPG1 and SIGMAR1 for organelle integrity and bioenergetic capacity. Together, our findings establish Contact-phagy as a selective pathway for the turnover of defined organelle contact site subdomains and suggest a broader principle by which interorganelle interfaces are maintained.

cell biology↗

HUH-tag conjugation enables accessible, site-specific functionalization of DNA origami with signaling-competent proteins

DNA origami enables precise nanometer scale control over the number, spacing, and geometry of biomolecules, making it a powerful tool for dissecting spatially organized biological processes such as receptor signaling. However, functionalizing origami with proteins remains a bottleneck restricting the technology to specialized laboratories. Here we show that viral-derived HUH endonuclease tags, small (~10 kDa) domains that form a covalent phosphotyrosine bond with short unmodified ssDNA, provide a simple and cost-effective route to decorating DNA origami with functional proteins. Using interleukin-1{beta} (IL-1{beta}) as a model ligand, we generated recombinant IL-1{beta}-HUH, achieved efficient site-specific conjugation to unmodified ssDNA within minutes, and coupled the conjugates to a DNA origami pegboard with tunable stoichiometry. IL-1{beta}-functionalized origami triggered ligand-dependent signalling in living cells and allowed visualization of the recruitment of downstream signaling molecules, confirming that HUH-tag conjugation preserves functionality. Because this strategy requires only standard molecular biology equipment and unmodified DNA, it lowers a technical and financial barrier to DNA origami-based experimentation, opening the approach to a broader range of biological questions and laboratories outside the DNA nanotechnology field.

cell biology↗

Multivalent BBSome-IFT proximities support IFT-A-IFT-B coordination in vivo

Intraflagellar transport (IFT) depends on coordinated movement of IFT-A and IFT-B, yet how these assemblies remain coupled within dynamic ciliary trains is unclear. Here, we combine endogenous fluorescent knock-in tagging, quantitative in vivo FLIM-FRET, structural modeling, and targeted mutagenesis to define IFT organization in C. elegans and mammalian cells. FLIM-FRET mapping identifies proximity networks within IFT-B and places IFT144 near IFT172 and IFT88 in intact cilia. Loss of BBSome function is associated with weaker IFT-A-IFT-B proximity signals and divergent subcomplex transport dynamics, consistent with a contribution of the BBSome to IFT coordination in vivo. Split-GFP complementation places BBSome components near IFT-A and IFT-B, while structural modeling nominates complementary charged surfaces on the IFT46-IFT52 dimer and BBS7/BBS9. Charge-reversal mutations targeting these surfaces impair BBSome recruitment and functional rescue. Together, these findings support a model in which multivalent BBSome-IFT proximities contribute to IFT-A-IFT-B coordination during ciliary transport.

cell biology↗