Biology subjects

Chiduza, G. N.

Publications and source records attributed to Chiduza, G. N..

3 recordsLinked to original sources

bioRxiv2025

Proposed mechanism for Rft1-mediated scrambling of a dolichol-linked oligosaccharide

The membrane protein Rft1 is proposed to play an essential role in yeast and human cells by scrambling the glycolipid Man5GlcNAc2-PP-dolichol (M5-DLO) across the endoplasmic reticulum (ER) for protein N-glycosylation. While this activity has been demonstrated in liposomes reconstituted with purified Rft1, biochemical evidence of additional M5-DLO scramblases and the viability of Rft1-null Trypanosoma brucei suggest that scrambling may be a moonlighting function of Rft1 rather than its essential cellular role. To investigate this paradox, we used AlphaFold3 and Chai-1 to model the conformational dynamics of yeast Rft1-M5-DLO complexes. The models suggest an alternating access mechanism, typical of Multidrug/Oligosaccharidyl-lipid/Polysaccharide (MOP) superfamily transporters, in which a cationic central cavity coordinates the anionic headgroup of M5-DLO, while the dolichol tail of the lipid is accommodated through a lateral portal formed by two transmembrane helices. We used the models to design mutations to disrupt the interaction between Rft1 and the M5-DLO headgroup, and to engineer a salt bridge to block the portal and stall transport. Using a Tet-off yeast reporter strain, we tested 26 central cavity mutants and identified two that supported cell growth poorly despite being well-expressed. Strikingly, the portal-blocking mutant which lacks scramblase activity supported robust growth. These data suggest that while M5-DLO binding is important for Rft1s essential function, scrambling activity is dispensable. We propose that Rft1s essential role may be as an M5-DLO chaperone, capturing and routing M5-DLO propitiously on the cytoplasmic side of the ER to coordinate DLO biosynthesis. ImportanceCell surface and secreted proteins are decorated with sugar chains. These chains are first assembled on a lipid carrier. Initial stages of assembly occur on the cytoplasmic side of a subcellular structure called the endoplasmic reticulum (ER). To complete assembly, the partially assembled lipid-linked sugar chain must be flipped across the ER. Here we use computationally guided cell-based assays to examine the role of the Rft1 protein in this process.

biochemistry↗

bioRxiv2024

Molecular characterization of Rft1, a membrane protein associated with congenital disorder of glycosylation type 1N

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1s ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

cell biology↗

bioRxiv2023

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically de-acidified compartments targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to pH gradient disruption is unclear. Here, we reveal a direct role for the V1H subunit of the V-ATPase proton pump in recruiting ATG16L1. The interaction between V1H and ATG16L1 occurs within assembled V-ATPases, but not dissociated V1 complexes. This selectivity allows recruitment to be coupled to changes in V-ATPase assembly that follow pH dissipation. Cells lacking V1H undergo canonical macroautophagy but are unable to recruit ATG16L1 in response to influenza infection or ionophore drugs. We identify a loop within V1H that mediates ATG16L1 binding, which is absent in a neuronal isoform of V1H. Thus, V1H controls ATG16L1 recruitment in response to proton gradient dissipation, suggesting that the V-ATPase acts autonomously as a cell-intrinsic damage sensor.

molecular biology↗