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Assay of genes involved in GPI biosynthesis in ER ~Partitioning of GPI-anchored proteins with Triton X-114
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Assay of genes involved in GPI biosynthesis in ER ~Partitioning of GPI-anchored proteins with Triton X-114

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Category
GPI anchored proteins
Protocol Name

Assay of genes involved in GPI biosynthesis in ER ~Partitioning of GPI-anchored proteins with Triton X-114

Authors
Fujita, Morihisa
Research Institute for Microbial Diseases, Osaka University

Maeda, Yusuke
Research Institute for Microbial Diseases, Osaka University

Kinoshita, Taroh *
Research Institute for Microbial Diseases, Osaka University
*To whom correspondence should be addressed.
KeyWords
Reagents

Triton X-114

Phosphate-buffered saline (PBS)

Phosphatidylinositol-specific phospholipase C (PI-PLC)

Suspension buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, Protease inhibitor cocktail)

PI-PLC reaction buffer (20 mM Tris-HCl (pH 7.4), 0.1% Triton X-100, Protease inhibitor cocktail)

Instruments

Incubator (37˚C), centrifuge

Methods
1.

Preparation of 12% Triton X-114 solution

1) 

 Dissolve 1.5 g Triton X-114 in 50 mL PBS on ice (or in cold room).

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2) 

 Warm the solution at 37˚C until the solution becomes turbid.

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3) 

 Centrifuge at 1000 × g for 10 min at room temperature.

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4) 

 Remove and discard the upper phase (aqueous phase).

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5) 

 Redissolve the lower phase (detergent phase) in an equal volume of cold-PBS and mix on ice.

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6) 

 Repeat 2) to 5) three times.

(The final detergent phase contains around 12% Triton X-114.)

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7) 

 Store at 4˚C until further use.

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2.

Partitioning of GPI-anchored proteins with Triton X-114

1) 

 Suspend cells (2 × 106) in 1 mL PBS.

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2) 

 Centrifuge at 300 × g for 3 min and resuspend pellet in 100 μL Suspension buffer.

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3) 

 Add 20 μL of 12% Triton X-114 solution (2% final concentration), vortex well and incubate on ice for 15 min.

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4) 

 Centrifuge at 13,000 × g for 15 min at 4˚C and transfer supernatant to a fresh tube.

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5) 

 Add 8-fold volume of ice-cold acetone and incubate for 1 h at −80˚C.

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6) 

 Centrifuge at 13,000 × g for 15 min at 4˚C, discard supernatant and dry protein pellet.

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7) 

 Resuspend protein pellet in 200 μL PI-PLC reaction buffer.

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8) 

 Divide suspension into two and add PI-PLC (final: 1 unit/mL) or 50% glycerol (control).

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9) 

 Incubate at 37˚C for 1 h.

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10) 

 Add 20 μL of 12% Triton X-114 solution (2% final concentration), vortex well and incubate on ice for 10 min.

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11) 

 Centrifuge at 1,000 × g for 10 min at 37˚C (alternatively, use a compact centrifuge such as Chibitan (Millipore, 5,600 × g for 7 min) in incubator at 37˚C).

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12) 

 Transfer the upper phase to a fresh tube (upper phase: aqueous phase, lower phase: detergent phase).

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13) 

 Add 100 μL warmed PI-PLC reaction buffer to the lower phase, incubate at 37˚C for 2 min and remove the upper phase by centrifugation at 1,000 × g for 10 min at 37˚C (wash detergent phase to remove contaminants from aqueous phase).

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14) 

 Add 20 μL Triton X-114 to aqueous phase (step 12), add 100 μL PI-PLC reaction buffer to detergent phase (step 13) (to keep sample conditions and volumes consistent).

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15) 

 Analyze by western blotting. If necessary, immunoprecipitate proteins of interest prior to western blotting.

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Notes

Inositol-acylated GPI anchors are not cleaved by PI-PLC. During GPI biosynthesis in the endoplasmic reticulum (ER), an acyl chain is transferred to the 2-position of inositol on GPI intermediately. Therefore, GPI intermediates are resistant to PI-PLC. Soon after GPI attachment to proteins, the acyl chain is usually eliminated by GPI-inositol deacylase PGAP1 in the ER. The deacylated GPI anchors then become sensitive to PI-PLC. Therefore, GPI-anchored proteins on mammalian cells are usually sensitive to PI-PLC. However, GPI-anchored proteins on human and mouse erythrocytes are resistant to PI-PLC treatment because the GPI structures maintain the acyl chain on the 2 position of the inositol.

Figure & Legends

Figure & Legends

Fig. 1. PI-PLC sensitivity of extracted FLAG-tagged CD59.

CHO-K1 cells were transiently transfected with FLAG-tagged CD59 cDNAs, treated with (+) or without (−) PI-PLC, lysed in 2% Triton X-114 on ice, and partitioned into the aqueous (A) and detergent (D) phases. FLAG-tagged CD59 proteins were immunoprecipitated from the detergent and aqueous phases with anti-FLAG beads and analyzed by SDS-PAGE/western blotting under reducing conditions using an anti-FLAG antibody.

This figure was originally published in J Biol Chem. Tanaka S. et al. "Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p" 2004, 279(14):14256–63. © the American Society for Biochemistry and Molecular Biology.

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