Protein complex identification by GFP-transgene proteomics:

Arabidopsis Retinoblastoma-related (RBR) and E2F proteins;

less canonical but more complex interactions
Aladár Pettkó-Szandtner1, Zsuzsa Darula1, László Bögre2, Zoltán Magyar3 and Katalin F. Medzihradszky1
1
Laboratory of Proteomic Research, Biological Research Centre, Szeged, Hungary; 2Royal Holloway, School of Biological Sciences, University of London, Egham, Surrey, UK; 3Institute of Plant Biology,
Biological Research Centre, Szeged, Hungary

E2FB core complex interactions, DREAM complex and axillary partners.

Summary
Cell cycle-regulated gene expression is highly coordinated events throughout the cell cycle. The conserved multiprotein
p35S:FBL17-GFP
DP, RB, E2F and multi-vulval class B (DREAM) complex provides a unifying role in the cell cycle by directly linking animal
pocket proteins, transcription factor E2F, BMYB and forkhead box protein M1. DREAM complexes have general roles as Input GFP-IP

a global repressor and Myb transcription factors counteract the repression (1). Although plants share the conserved A B C 1/50
N T N T
members of E2F, DP, RB, Myb proteins (2;3), and some MuvB components, the existence of DREAM repressor complex
and its possible functions during cell proliferations are not known. By using various biochemical approaches, such as P-RBR

co-immunoprecipitation and quantitative transgene–green fluorescent protein interactomics (modified QUBIC, 4) we

have identified two different DREAM-related complexes from the developing first leaf pairs of transgenic Arabidopsis RBR

carrying the GFP-tagged version of MYB or E2F or RBR1 genomic clones.

E2FB
LC-MS/MS analysis of immunoprecipitated GFP-tagged MYB3R3, RBR1, and E2FB revealed an association of repressor Uniprot ACC# Protein name Unique peptide
MYB3R3 with the LIN9 orthologs ALY2 and ALY3 and the LIN54 ortholog TCX5. The activator E2FB was found to E2FA
Q8W104 F-box/LRR-repeat protein 17 36
associate with ALY3 and TCX5 in addition to RBR1, DPA, and DPB. The co-immunoprecipitations in early and late stages
of leaf development show that the repressor MYB3R3 did not bind to the activator E2FB but instead binds to RBR1 and Q9FV71 Transcription factor E2FB 5 FBL17-GFP
the repressor E2FC at later stages of leaf development when cell proliferation rate is reduced. In contrast, the activator Q9FHW7 SKP1-like protein 1B/AT5G42190 2
MYB3R4 binds to activator E2FB and RBR1 but not to repressor E2FC in early proliferating stages of leaf development. DPA
Q39255 SKP1-like protein 1A/AT1G75950 2
Thus, Arabidopsis appears to employ two distinct complexes for transcriptional regulation of G2/M genes that are
temporarily separated during development. (5, 6) Transcription factor-like protein CDKA;1
Q38820 DPB 1
1. Sadasivam S, DeCaprio JA. Nat Rev Cancer. 2013, 3(8):585-95.
2. Vandepoele K, et al. Plant Cell. 2002, (4):903-16. Loading
control
3. Ito M, J Plant Res 2005 118: 61–69.
4. Hubner NC, et al. J Cell Biol. 2010189(4):739-54.
5. Kobayashi et al., EMBO J. 2015
6. Horvath MB et al., EMBO J. 2017 (A)According to the meta-analyzes of 26 different E2FB IPs we were able to distinguish between different complexes.
Regardless of the applied treatments the E2FB was co-purified in stoichiometric amount with its best studied partners (RBR1,
MYB3R3 and MYB3R4 both interact with RBR1 and differentially associate with E2F isoforms DPA/B). The ratio between DREAM complex members and the E2FB bait were significantly lower. We were also able to detect
several axillary interactions, these amount and detectability depends on the used treatments (age, stress, day length etc.)
Unit=1/ln(specificity) specificity=relative abundance in sample/relative abundance in promiscuome * quality control corrected
detection frequency.
The interaction between E2FB and FBL17 was further confirmed in additional pull-down experiments using transgenic
Arabidopsis plants expressing FBL17-GFP under the control of a strong viral promoter p35S. GFP pull downs were further
analysed either in MS (B) or in immuno-blott assays (C).

The E2FB transcription factor is not just nuclear localised but seems to present in the plasma
membrane. E2FB interacts with PIN proteins

A B

PIN3-GFP

PIN3-GFP

p35S-GFP
Input 1/50

IP-GFP

IP-GFP
E2FB

pE2FA:gE2FA-3xvYFP Loading
control

GFP
(ponceau)

pE2FB:gE2FB-3xvYFP

Cell cycle activator E2FB transcription factor was detected in cell boundaries of transgenic Arabidopsis leaf expressing E2FB in
fusion with triple Venus-tag (3xvYFP) (green signal) under the control of its own promoter. In contrast, closest relative E2FA
was predominantly nuclear localised in pE2FA:gE2FA-3xvYFP transgenic leaf. Confocal laser microscopy images were taken
after propidium iodide staining (red signal)(A). PIN3-GFP expressing plants were used to immunoprecipitate (IP) protein
complexes through the GFP epitope, and the presence of E2FB was detected by using E2FB specific antibody in immunoblott
assay(B).

Detected Arabidopsis RBR1 in vivo phosphosites (isolated through E2FB/E2FC)

Phospho@9
MYB3R3 and MYB3R4 both interact with RBR1 and differentially associate with E2F isoforms
Phospho@374|375
Phospho@385
A, B MYB3R3‐GFP and GFP‐MYB3R4 both interact with RBR1 and CDKA;1, but with a different E2F isoform in Arabidopsis leaves. Phospho@389
IP was performed with anti‐GFP antibodies from protein extracts prepared from first leaf pairs of MYB3R3‐GFP or GFP‐MYB3R4 Phospho@406
transgenic plants at indicated days after germination (DAG). In these transgenic plants, expression of GFP fusion proteins was Phospho@685
driven by the corresponding native promoters. Co‐IP of RBR1 and E2FB was examined by Western blot analyses using Phospho@708
corresponding antibodies. For detection of MYB3R3‐GFP and CDKA;1, anti‐GFP and anti‐PSTAIRE (specific to CDKA;1) Phospho@712|714
Phospho@885
antibodies were used. As input, 1/10 of IP was loaded. Coomassie staining of the same membrane was used as a loading control.
Phospho@898
Phospho@911
C. MYB3R3‐GFP interacts with E2FC, but GFP‐MYB3R4 does not. IP was performed with anti‐GFP antibodies from protein Phospho@936|937|940|942
extracts prepared from first leaf pairs of MYB3R3‐GFP or GFP‐MYB3R4 transgenic plants at indicated days after germination Phospho@940|942
(DAG). Co‐IP of E2FC and CDKA;1 was examined by Western blot analyses using anti‐E2FC and anti‐PSTAIRE antibodies, Phospho@936|942
respectively. As input, 1/16 of IP was loaded. Coomassie staining of the same membrane was used as a loading control. Phospho@942

Several phosphopeptides were identified by MS/MS from RBR1-GFP overexpressing plants, direct RBR1-GFP pull downs (RBR1
as bait) and from E2Fs-GFP pull downs (E2F bound, prey RBR1). Not surprisingly the phosphosites of the Arabidopsis RBR1 show
QUBIC workflow in label-free format strong similarities to the human ortholog. Interestingly, although the E2F bound RBR1 fraction contains several phosphosites,
the well studied S911( )phosphorylation, which is known to prevent E2F binding was present only in the free RBR1 fraction.

Suggested animal and Arabidopsis Dream-complex model by J. A. DeCaprio

DREAM-complex interaction partners of E2FB, RBR1 and MYB3R3

Peptide counts of label free pull downs of

UNIPROT Name of identified interacting DREAM complex

ACC# proteins AGI number E2FA E2FB E2FC RBR1 DPA DPB MYB3R3

Q9LKZ3 RBR1 Retinoblastoma-related protein 1 AT3G12280 54 59 54 310 21 30 0

Q9FV71 E2FB Transcription factor E2FB AT5G22220 0 47 0 16 30 15 0

Q9FNY2 DPB Transcription factor-like protein DPB AT5G03415 27 29 26 15 0 68 0

Q9FNY3 DPA Transcription factor-like protein DPA AT5G02470 9 10 6 0 41 0 0

Q9FV70 E2FC Transcription factor E2FC AT1G47870 0 0 81 0 10 4 0

Q9FNY0 E2FA Transcription factor E2FA AT2G36010 36 0 0 0 10 4 0

Q6A332 ALY3 Protein ALWAYS EARLY 3 AT3G21430 0 5 9 0 6 8 2

O22467 MSI1 Histone-binding protein MSI1 AT5G58230 0 4 8 0 4 5 0

Q9SZD1 TCX5 Protein tesmin/TSO1-like CXC 5 AT4G29000 0 4 5 1 4 0 4

Q6A333 ALY2 Protein ALWAYS EARLY 2 AT3G05380 0 2 4 0 0 0 4

Distinct MYB activator and repressor complexes in Arabidopsis thaliana.
Q9S7G7 MYB3R-1 Myb-related protein 3R-1 At4g32730 0 1 2 0 0 0 0 Distinct MYB activator and repressor complexes regulate G2/M gene expression in Arabidopsis proliferating and post-mitotic
endocycling cells. MYB3R3/4 may bind directly to MSA elements and E2FB/C to E2F elements. It is not known whether TCX5, the
Q9SL70 TCX6 Protein tesmin/TSO1-like CXC 6 At2g20110 0 1 0 0 0 0 0
Arabidopsis ortholog of LIN54, binds directly to DNA.

DREAM-complex interaction partners of E2FA, E2FB, E2FC, RBR1, DPA, DPB and MYB3R3
Seedlings (7 DAG) expressing GFP tagged E2FA/B/C, RBR1, DPA/B and MYB3R3 under their own promoters were collected and
Bolyai Research Scholarship of the Hungarian Academy of Sciences
used for separate immunoprecipitation experiments. Immuno Purified samples were analyzed by LC MS/MS Numbers indicate
given to A.P.‐Sz., Hungarian Scientific Research Found (OTKA
the number of identified unique peptides for the respective proteins. 105816) given to Z.M.
S4). Interestingly, in the E2FA-GFP pull downs we could never detect any of the components of the multi-protein DREAM The research was also supported by the Economic Development and
complex (DP, RB-like E2F, and MuvB, (1), while with E2FB-GFP, these proteins were readily Innovation Operation Programme (GINOP-2.3.2-15-2016-00032).
pull down. This may suggest that E2FA functions in different complex(es) (6) than the DREAM associated with E2FB and E2FC
(5). None of these proteins were identified when the GFP-expressing control plants were analyzed.