1. Bert Rutten Aurin Vos Sacha Dalhuijsen Supervisor: Eefjan Breukink
Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein Jonne Helenius*, Davis T. W. Ng², Cristina L. Marolda³, Peter Walter§, Miguel A. Valvano³ & Markus Aebi*
Bert Rutten
Aurin Vos
Sacha Dalhuijsen
Supervisor: Eefjan Breukink

2. Introduction Proteins in eukaryotic cells are glycosylated
Pathway N-linked glycolysation is highly conserved
Biosynthesis of N-linked oligosacharides requires modification both outside as inside the ER
Proteins in Eukaryotic cells are N-glycosylated. This is to protect proteins from enzymatic cleavage, and in some cases nesessary for the function of the protein.
The pathway witch produces the sugarchains is a highly conserved pathway. Important in this article is that N-linked olichosacharides require modification in both the cytosolic part of the ER as inside the ER.

3. Introduction Man5GlcNAc2-PP-Dol intermediate in cytosolic side
Absence GDP-Man within ER
Modification continues on the lumenal side
ConcanavalinA (mannose-specific-lectin) only binds Man5 and smaller in assay
Man5GlcNac-PP-Dol flipped by flippase
That N-linked oligosacharides biosynthesis requires modification both at the cytosolic side of the ER as in the ER lumen is shown in this figure.

4. Introduction
Search for the flippase in Saccharomyces cerevisiae (Yeast)
Mutant deficient in N-linked glycosylation
Mutation in Rft1
Haploid strain with Rft1 under Gal1-10 promotor
Growth on glucose represses Rft1

5. Rft1 mutant Glycosylation effects measured on CPY
4 glycosylation sites
high-performance liquid chromatography (HPLC)
Labelling with [3H] mannose
A) Westernblot analysis of CPY
B) high-performance liquid chromatography (HPLC)

6. Rft1mutant Three explanations Deficiency Alg3 Decreased substrate Alg3
No flippase

7. 1) Deficiency Alg3 In alg3 Man5 is transferred to protein
Proteins in alg3 are resistant to EndoH digestions
Glycosylated CPY in Rft1 mutant is sensitive to EndoH digestion
No Alg3 deficiency

8. 2) Decreased substrate Alg3
dpm1-6 deficient in Dol-P-Man synthase
Altered O- and N-linked glycosylation
Rft1 no altered O-glycosylation
No decrease in Dol-P-Man

9. Rft1mutant
Deficiency Alg3
Decreased substrate Alg3
No flippase

10. 3) No flippase Is Rft1 a flippase? Man3 is inefficiently translocated
alg11 mutant
Alg11 is required for Man5 synthesis
Accumulate Man3
Man3 is inefficiently translocated
Alg3 adds a Man
truncated olichosacharide

11. 3) No flippase alg11  accumulation Man3 and Man7
pRFT1 in alg11 restores Man7 production

12. 3) No flippase
It seems that there are more sites glycosylated in the alg11 strain with pRFT1 overexpression

13. Conclusions
Rft1 is required for membrane translocation of Man5GlcNAc2

14. Does Rft1 flip an N-glycan lipid precursor. Arising from: J
Does Rft1 flip an N-glycan lipid precursor? Arising from: J. Helenius et al. Nature 415, 447–450 (2002)
Biochemical study: Rft1 no flippase activity!

15. Flippase assay Vesicles with labelled Man5 ConA binds Man5 on outside
50% of Man5
Vesicles with Man5 and ER membrane proteins (TE)
ConA binding prevents flipping outside inside
Flippase flips inside  outside
~100% Man5

16. Flippase assay Dose-response plots
Measure amount of ConA bound Man5 with different amounts of ER proteins
Slope determines flippase abundance

17. Role of RFT1 as flippase GAL1-10 Rft1
Rft1 has same flippase abundance as WT
TE depleted Protein A tagged Rft1 with IgG beads
ER membrane proteins with/without Rtf1 has same flippase abundance

18. Role of RFT1 as flippase TE fractionated by CM-sepharose
Rft1 abundance does not correlate with flippase activity

19. Role of RFT1 as flippase
TE fractionated by velocity gradient sedimentation
Rft1 abundance does not correlate with flippase activity

20. Conclusions Rft1 is not the flippase
Rft1 may act in recruitment of Man5GlcNAc-PP-Dol to the flippase

21. Discussion
In vivo
Based on genetics, not confirmed with biochemical assay.
Authors do not prove direct function as flippase Rft1
In vitro
Using triton gives denatured membrane proteins and perturb membrane structures
Not all flippases give flippase activity
Abundance might not correlate with activity
 Conformation of genetics with biochemical study

22. Future experiments Ratio cytosolic/lumenal oligosaccharides
Testing IgG extracted Rft1 flippase activity
Positive control flippase assay with known flippase