Many eukaryotic cell surface proteins with various functions are anchored to the membrane via glycosylphosphatidylinositol (GPI). Triton X-114 is a non-ionic detergent with a low cloud point of 23˚C; an indicator for phase separation of hydrophilic proteins from amphiphilic proteins. At temperatures above the cloud point, detergent solutions separate into aqueous and detergent-enriched phases. GPI-anchored proteins are usually separated into detergent phase. Once GPI moieties are cleaved by enzymes including PI-PLC and GPI-PLD, the protein parts no longer partition into the detergent phase and recover into the aqueous phase. This characteristic of partitioning behavior is useful when the GPI anchor is present. Here, we describe a protocol for partitioning of GPI-anchored proteins with Triton X-114 combined with PI-PLC treatment. |
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
|
● |
|
● |
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). |
Comment 0
|
|
2) |
Warm the solution at 37˚C until the solution becomes turbid. |
Comment 0
|
|
3) |
Centrifuge at 1000 × g for 10 min at room temperature. |
Comment 0
|
|
4) |
Remove and discard the upper phase (aqueous phase). |
Comment 0
|
|
5) |
Redissolve the lower phase (detergent phase) in an equal volume of cold-PBS and mix on ice. |
Comment 0
|
|
6) |
Repeat 2) to 5) three times.
(The final detergent phase contains around 12% Triton X-114.) |
Comment 0
|
|
|
|
2. |
Partitioning of GPI-anchored proteins with Triton X-114
|
1) |
Suspend cells (2 × 106) in 1 mL PBS. |
Comment 0
|
|
2) |
Centrifuge at 300 × g for 3 min and resuspend pellet in 100 μL Suspension buffer. |
Comment 0
|
|
3) |
Add 20 μL of 12% Triton X-114 solution (2% final concentration), vortex well and incubate on ice for 15 min. |
Comment 0
|
|
4) |
Centrifuge at 13,000 × g for 15 min at 4˚C and transfer supernatant to a fresh tube. |
Comment 0
|
|
5) |
Add 8-fold volume of ice-cold acetone and incubate for 1 h at −80˚C. |
Comment 0
|
|
6) |
Centrifuge at 13,000 × g for 15 min at 4˚C, discard supernatant and dry protein pellet. |
Comment 0
|
|
7) |
Resuspend protein pellet in 200 μL PI-PLC reaction buffer. |
Comment 0
|
|
8) |
Divide suspension into two and add PI-PLC (final: 1 unit/mL) or 50% glycerol (control). |
Comment 0
|
|
10) |
Add 20 μL of 12% Triton X-114 solution (2% final concentration), vortex well and incubate on ice for 10 min. |
Comment 0
|
|
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). |
Comment 0
|
|
12) |
Transfer the upper phase to a fresh tube (upper phase: aqueous phase, lower phase: detergent phase). |
Comment 0
|
|
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). |
Comment 0
|
|
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). |
Comment 0
|
|
15) |
Analyze by western blotting. If necessary, immunoprecipitate proteins of interest prior to western blotting. |
Comment 0
|
|
|
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. |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
Date of registration:2015-01-07 14:15:38 |
- Tanaka, S., Maeda, Y., Tashima, Y. and Kinoshita, T. (2004) Inositol-deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. J. Biol. Chem. 279, 14256–14263 [PMID : 14734546]
- Doering, T. L., Englund, P.T., and Hart, G.W. (2001) Detection of Glycophospholipid Anchors on Proteins. Current Protocols in Molecular Biology 17.8.1–17.8.13 [PMID : 18265164]
|
This work is licensed under Creative Commons Attribution-Non-Commercial Share Alike. Please include the following citation
How to Cite this Work in an article:
Fujita, Morihisa,
Maeda, Yusuke,
Kinoshita, Taroh,
(2015). GlycoPOD https://jcggdb.jp/GlycoPOD.
Web.29,3,2024 .
How to Cite this Work in Website:
Fujita, Morihisa,
Maeda, Yusuke,
Kinoshita, Taroh,
(2015).
Assay of genes involved in GPI biosynthesis in ER ~Partitioning of GPI-anchored proteins with Triton X-114.
Retrieved 29,3,2024 ,
from https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t76.
html source
Fujita, Morihisa,
Maeda, Yusuke,
Kinoshita, Taroh,
(2015).
<b>Assay of genes involved in GPI biosynthesis in ER ~Partitioning of GPI-anchored proteins with Triton X-114</b>.
Retrieved 3 29,2024 ,
from <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t76" target="_blank">https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t76</a>.
Including references that appeared in the References tab in your work is
much appreciated.
For those who wish to reuse the figures/tables, please contact JCGGDB
management office (jcggdb-ml@aist.go.jp).
|
|