RFT1 affects GPI anchor glycosylation.

Gottier, Petra; González Salgado, Amaia; Menon, Anant K; Liu, Yuk-Chien; Acosta-Serrano, Alvaro; Bütikofer, Peter (2017). RFT1 affects GPI anchor glycosylation. Journal of biological chemistry, 292(3), pp. 1103-1111. American Society for Biochemistry and Molecular Biology 10.1074/jbc.M116.758367

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The membrane protein RFT1 is essential for normal protein N‑glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid Man5GlcNAc2-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N‑glycosylation but also glycosylphosphatidylinositol (GPI) anchor side chain modification. Analysis by immunoblotting, metabolic labeling and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites compared to wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Gottier, Petra; González Salgado, Amaia and Bütikofer, Peter

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

19 Apr 2017 09:06

Last Modified:

21 Jan 2018 02:30

Publisher DOI:

10.1074/jbc.M116.758367

PubMed ID:

27927990

Uncontrolled Keywords:

Golgi; N-linked glycosylation; Trypanosoma brucei; endoplasmic reticulum (ER); glycosylphosphatidylinositol (GPI anchor)

BORIS DOI:

10.7892/boris.93990

URI:

https://boris.unibe.ch/id/eprint/93990

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