Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

Jonne Helenius*, Davis T. W. Ng ², Cristina L. Marolda³, Peter Walter §, Miguel A. Valvano ³ & Markus Aebi* Bert Rutten Aurin Vos Sacha Dalhuijsen Supervisor: Eefjan Breukink

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

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

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

Rft1 mutant

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

Rft1mutant

• Three explanations 1) Deficiency Alg3 2) Decreased substrate Alg3 3) No flippase

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

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

Rft1mutant

1) Deficiency Alg3 2) Decreased substrate Alg3 3) No flippase

3) No flippase

• Is Rft1 a flippase?

–

alg11

mutant • Alg11 is required for Man5 synthesis • Accumulate Man3 • Man3 is inefficiently translocated – Alg3 adds a Man • truncated olichosacharide

3) No flippase

•

alg11

 accumulation Man3 and Man7 • pRFT1 in

alg11

restores Man7 production

3) No flippase

• It seems that there are more sites glycosylated in the

alg11

strain with pRFT1 overexpression

Conclusions

• Rft1 is required for membrane translocation of Man5GlcNAc2

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!

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

Flippase assay

Dose-response plots Measure amount of ConA bound Man5 with different amounts of ER proteins  Slope determines flippase abundance

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

Role of RFT1 as flippase

TE fractionated by CM-sepharose  Rft1 abundance does not correlate with flippase activity

Role of RFT1 as flippase

TE fractionated by velocity gradient sedimentation  Rft1 abundance does not correlate with flippase activity

Conclusions

• Rft1 is not the flippase • Rft1 may act in recruitment of Man5GlcNAc-PP-Dol to the flippase

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

Future experiments

• Ratio cytosolic/lumenal oligosaccharides • Testing IgG extracted Rft1 flippase activity • Positive control flippase assay with known flippase