Final Report Summary - TGNPROTEOMICS (Proteomics of the trans-Golgi network in Arabidopsis thaliana using immunoisolation as a means of organelle purification)
Publishable summary
The overall aim of TGNPROTEOMICS is to identify novel proteins in the Arabidopsis thaliana Trans-Golgi network (TGN), a poorly characterised organelle. The TGN is known to play an important role in sorting and forward transport of proteins leaving the Golgi apparatus, but also in retrograde transport and endocytosis. The knowledge generated in this project will help understanding cellular transport in plants. This proposal involves the development of a purification strategy for TGN membranes that can also be applied to isolate other compartments from plants. The TGN membranes will be purified from a line expressing a green fluorescent protein (GFP) fusion of the TGN-specific H+-ATPase subunit VHA-a1 (VHA-a1-GFP) throughout the plant. A TGN-enriched fraction will be obtained by density centrifugation of a complete lysate and this fraction will be used to immuno-purify TGN membranes using GFP antibodies immobilised on magnetic beads. The peptide content of the purified TGN will be determined by liquid chromatography-mass spectrometry (LC-MS/MS). The most interesting proteins of those identified in the TGN will be characterised further.
The objectives for the first period (one year) included setting up:
-1. A system for growth of Arabidopsis seedlings in sterile conditions, with high root biomass. One of the requirements was that the seedlings were easily purified without agar or soil contaminants;
-2. Methods for cell fractionation in order to obtain sufficient enrichment of TGN membranes, with only small amounts of chloroplast and mitochondrial membranes;
-3. A protocol for the binding of VHA-a1-GFP to a solid phase and affinity purification;
-4. generating constructs to provide for a back-up system using alternative tags to GFP
With regard to objective 1, a system was developed in which seedlings were grown in flasks in liquid medium with very low levels of agar. The purity and yield of the biomass generated in this way was satisfactory. It was possible to separate the shoot from the root area of the seedling, something that was also a condition for subsequent procedures. Methods for cell fractionation (objective 2) were derived partly from protocols developed in the laboratory of Professor Kathryn Lilley at the Cambridge Centre for Proteomics (CCP). The density centrifugation used in this laboratory was not fully implemented as part of our cell fractionation method, as it was deemed not necessary to achieve this level of purity before affinity purification. The final method included homogenisation in a Hepes-sucrose buffer, two centrifugation steps, followed by a concentration step on an 'iodixanol cushion', a simplified form of density centrifugation, in order to concentrate the cell membranes. The resulting 'membrane fraction' was the starting point for affinity purification. Objective 3 required consideration of different affinity purification systems. A single-chain GFP antibody immobilised on sepharose, the so-called GFP-trap, was an important find. This camel antibody is more specific than most GFP antibodies and binds with very high affinity (binding strength). A strategy was developed to purify VHA-a1-GFP from the membrane fraction. The number of contaminating proteins was minimal but yields were low. Unfortunately, purification needed low levels of detergent (0.01% Igepal or 3 mM digitonin). A preliminary mass spectrometry analysis confirmed that the eluate was highly pure and that co-purification of additional subunits of H+-ATPase was successful. Future work will focus on extending purification of the complex to purification of intact TGN membranes.
GFP and yellow fluorescent protein (YFP) are universal tags in plant research because of their use in fluorescent microscopy. Using GFP-trap for purification of GFP- or YFP fusion proteins from plants can be applied widely to plant research and will simplify and accelerate purification of complexes for analysis by e.g. mass spectrometry.
The overall aim of TGNPROTEOMICS is to identify novel proteins in the Arabidopsis thaliana Trans-Golgi network (TGN), a poorly characterised organelle. The TGN is known to play an important role in sorting and forward transport of proteins leaving the Golgi apparatus, but also in retrograde transport and endocytosis. The knowledge generated in this project will help understanding cellular transport in plants. This proposal involves the development of a purification strategy for TGN membranes that can also be applied to isolate other compartments from plants. The TGN membranes will be purified from a line expressing a green fluorescent protein (GFP) fusion of the TGN-specific H+-ATPase subunit VHA-a1 (VHA-a1-GFP) throughout the plant. A TGN-enriched fraction will be obtained by density centrifugation of a complete lysate and this fraction will be used to immuno-purify TGN membranes using GFP antibodies immobilised on magnetic beads. The peptide content of the purified TGN will be determined by liquid chromatography-mass spectrometry (LC-MS/MS). The most interesting proteins of those identified in the TGN will be characterised further.
The objectives for the first period (one year) included setting up:
-1. A system for growth of Arabidopsis seedlings in sterile conditions, with high root biomass. One of the requirements was that the seedlings were easily purified without agar or soil contaminants;
-2. Methods for cell fractionation in order to obtain sufficient enrichment of TGN membranes, with only small amounts of chloroplast and mitochondrial membranes;
-3. A protocol for the binding of VHA-a1-GFP to a solid phase and affinity purification;
-4. generating constructs to provide for a back-up system using alternative tags to GFP
With regard to objective 1, a system was developed in which seedlings were grown in flasks in liquid medium with very low levels of agar. The purity and yield of the biomass generated in this way was satisfactory. It was possible to separate the shoot from the root area of the seedling, something that was also a condition for subsequent procedures. Methods for cell fractionation (objective 2) were derived partly from protocols developed in the laboratory of Professor Kathryn Lilley at the Cambridge Centre for Proteomics (CCP). The density centrifugation used in this laboratory was not fully implemented as part of our cell fractionation method, as it was deemed not necessary to achieve this level of purity before affinity purification. The final method included homogenisation in a Hepes-sucrose buffer, two centrifugation steps, followed by a concentration step on an 'iodixanol cushion', a simplified form of density centrifugation, in order to concentrate the cell membranes. The resulting 'membrane fraction' was the starting point for affinity purification. Objective 3 required consideration of different affinity purification systems. A single-chain GFP antibody immobilised on sepharose, the so-called GFP-trap, was an important find. This camel antibody is more specific than most GFP antibodies and binds with very high affinity (binding strength). A strategy was developed to purify VHA-a1-GFP from the membrane fraction. The number of contaminating proteins was minimal but yields were low. Unfortunately, purification needed low levels of detergent (0.01% Igepal or 3 mM digitonin). A preliminary mass spectrometry analysis confirmed that the eluate was highly pure and that co-purification of additional subunits of H+-ATPase was successful. Future work will focus on extending purification of the complex to purification of intact TGN membranes.
GFP and yellow fluorescent protein (YFP) are universal tags in plant research because of their use in fluorescent microscopy. Using GFP-trap for purification of GFP- or YFP fusion proteins from plants can be applied widely to plant research and will simplify and accelerate purification of complexes for analysis by e.g. mass spectrometry.