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bioRxiv · 10.1101/2024.04.03.587922

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

Hirata, E.·Sakata, K.-t.·Dearden, G. I.·Menon, I.·Chiduza, G. N.·Menon, A. K.
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Abstract

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.

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Hirata, E., Sakata, K.-t., Dearden, G. I., Menon, I., Chiduza, G. N., Menon, A. K.. 2024-04-03. Molecular characterization of Rft1, a membrane protein associated with congenital disorder of glycosylation type 1N. https://doi.org/10.1101/2024.04.03.587922

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