A novel strategy for the isolation of membrane ligand-receptor complexes
Perry Bateman, Ellie Marshall, Jose Gutierrez-Marcos
University of Warwick
Aims
•  To advance our knowledge about the role of receptor-ligand interactions in plants.
•  To develop a novel strategy for the isolation of low-abundance membrane protein
Experimental Approach
Results
•  I designed a strategy to enrich MEG13-BLRP from plant total protein extract.
•  Initial experiments using magnetic beads gave negative results on Western blot
•  This method is based on the strong protein-protein interaction between biotin and
streptavidin (SA) [3] as shown in figure 3.
complexes.
(figure 4.a). SA was eluted from the beads (MW: 52.8 kDa tetramer, 13.2 kDa
monomer, figure 4.b), and the antibody cross-reacted with the monomer. This was
indicated by the presence of the band in all lanes.
Introduction
1a 1b 1c •  In addition, lack of background bands in figure 4.b indicated that magnetic
streptavidin beads may not be suitable for our analysis.
•  Therefore, I compared the binding efficacy of different beads (figure 5). This
In plants, double fertilisation leads to the formation of a diploid embryo and a
analysis indicated that agarose beads were the more efficient.
triploid placental endosperm. The endosperm is thought to act only as a nourishing
organ to provide nutrients to the developing embryo. Surprisingly, we have found
that an Arabidopsis endosperm-specific protein, MEG13, is necessary for embryonic
development, acting specifically on the extra-embryonic suspensor cells [1]. Lossof-function analysis by RNA interference (RNAi) has revealed a wide range of
embryonic defects, in particular suspensor cell differentiation and embryo patterning
Total protein extracts from AtMeg13-BLRP (1a), Columbia (wild type) (1b) and mutant BLRP (1c)
plants were incubated with agarose-streptavidin beads. (Fig. 1). Interestingly, this protein is expressed exclusively in endosperm cells
surrounding the embryo, yet elicits a phenotype in suspensor cells. This suggests
that Meg13 acts non-autonomously as a diffusible signal between the endosperm
2 3 Figure 4. Western blot for AtMeg13 (a) and coomassie gel stain (b) of
eluted proteins from magnetic SA beads. Soluble proteins from WT
and Mutated BLRP: lanes (i) and (iii) respectively. Membrane bound
proteins from WT and Mutated BLRP: lanes (ii) and (iv) respectively.
50 pM of synthetic AtMeg13 as positive control (+ve).
and the embryo. However, it remains unclear how this protein can regulate these
developmental processes.
A B C D •  Total
soluble
proteins
were
extracted
with
Figure
5.
Coomassie
stained
proteins
eluted
from
magnetic
(a)
&
agarose (b & c) SA beads.
Background binding shown.
tris/triton
x-100
buffer,
Immunoprecipited with agarose beads but gave negative results after western blot
analysis (not shown). Therefore, I tested a different buffer to extract MEG13 from
different subcellular compartments as shown in figure 6.
Figure 1. Microscopy images of RNAi AtMeg13 phenotypes. Pro-embryo cells: yellow, suspensor
cells: red, the hypophysis: blue and the micropylar endosperm: green. Wild type (A) and RNAi
phenotypes; cone (B), double-head (C) and short suspensor (D).
The beads were pelleted and un-bound proteins washed away (2). The bound proteins were
eluted by boiling in SDS and centrifugation (3-4). 4 •  To address this caveat, we have generated a two-component system [2] designed
to Co-ImmunoPrecipitate (CoIP) the MEG13 protein complex.
•  To this aim, transgenic Arabidopsis plants were generated expressing an bacterial
Immunodetection
biotin ligase enzyme, BirA, and a MEG13 protein fusion to a Biotin Ligase
Figure 6. Western blot for
AtMeg13 in fractions of total
protein extract.
Sections (a),
(b) and (c) show cell wall bound,
soluble and microsomal fractions
respectively.
Sample
plants
carried the wild-type (WT) and
mutated (Mut) BLRP constructs.
50 pM of synthetic AtMeg13 as a
positive control (+ve).
Recognition Peptide (BLRP) as shown in figure 2. This system should biotinylate
MEG13 in vivo.
•  MEG13 was found, predominantly in the microsomal fraction, using a more
aggressive extraction buffer.
•  Co-expression of both
•  This indicated the protein was located in the membrane.
constructs during early
Key:
seed development was
confirmed by RT-PCR.
•  In addition, we
designed a MEG13
tagged with a biotin-
Meg13 Agarose beads bound to streptavidin
Meg13-BLRP
Biotin
Meg13-Mut BLRP Non-biotin binding proteins Figure 3. Schematic of biotin pull-down system.
defective BLRP tag by
engineering a key
•  Isolated peptides were detected by Western blotting using an antibody against
aminoacid (K->R)
MEG13.
substitution. •  Test experiments were carried out by spiking total protein extract from wild-type
Figure 2. Schematic of BLRP/BirA expression system.
plants with a synthetic biotin-tagged peptide (ZmMEG1).
Further Work
•  Confirm MEG13-BLRP abundance using an anti-BLRP antibody.
•  Repeat biotin extraction using the improved extraction procedure.
•  Increase scale of extraction to achieve higher yield of MEG13 and protein partners.
•  MS/MS analysis of isolated proteins.
References
1.  Bayer, M., et al., Paternal control of embryonic pa1erning in Arabidopsis thaliana. Science, 2009. 323(5920): p. 1485-­‐8. 2.  Law, J.A., et al., A protein complex required for polymerase V transcripts and RNA-­‐ directed DNA methyla@on in Arabidopsis. Curr Biol, 2010. 20(10): p. 951-­‐6 3.  Gonzalez, M., et al., Interac@on of bio@n with streptavidin. Thermostability and conforma@onal changes upon binding. J Biol Chem, 1997. 272(17): p. 11288-­‐94.