Transcript Analyses of the E3 ubiquitin-ligase target adapter

Analyses of the E3 ubiquitin-ligase target adapter-encoding BTB gene
families in algal species
Katie Plamann & Derek Gingerich
Department of Biology, University of Wisconsin—Eau Claire
Introduction
Ubiquitylation, the attachment of ubiquitin to proteins to mark for degradation by proteasomes, is crucial for proper
organism function. One family of complexes that play a role in this process is the BTB/Cullin 3/RBX E3 ubiquitinprotein ligases. Three proteins; a BTB (Bric-a-Brac, Tramtrack, Broad Complex) domain-containing protein, CUL3,
and RBX, form a complex to catalyze attachment of ubiquitin to target proteins (Fig. 1). The BTB proteins are the
target-adapters, binding to the proteins to be ubiquitylated via motifs appended to the BTB domain1.
Genes encoding BTB proteins have been identified in a wide range of eukaryotic organisms (including
fungi, protists, animals, and plants) but the BTB gene families in different groups show great variability in
size, complexity, and composition2. In land plant genomes thus far studied, BTB gene families are large (~75150 members) and complicated (with multiple subtypes based on the presence of a diverse set of encoded Figure 1. BTB/CUL3 E3
target-binding motifs)2. We are interested in when the particular BTB family composition seen in the higher ubiquitin-ligase complex
plants may have arisen in evolution.
To help answer this question, we have characterized the BTB gene families in the fully-sequenced genomes of the green
algal species Ostreococcus lucimarinus and Chlamydomonas reinhardtii. Aquatic green algae are thought to be the ancestors of
the land plants, so comparison of algal and land plant BTB families may give us clues as to how the BTB family evolved as the
transition to land occurred.
Identification of the BTB Gene Families in Genomes of
Chlamydomonas reinhardtii and Ostreococcus lucimarinus
Sequence Alignments of Ostreococcus lucimarinus and Representative
Chlamydomonas reinhardtii BTB Domains
Figure 3. Alignment of representative BTB domains from Chlamydomonas reinhardtii and
Ostreococcus lucimarinus. Predicted BTB domains from 7 Chlamydomonas BTB proteins and
the 2 Ostreococcus BTB proteins (4529 and 27814) were aligned with CLUSTALW2 using the
default settings. The alignment was displayed with BOXSHADE 3.21 using a 55% sequence
identity threshold. Conserved and similar amino acids are shown in black and gray boxes,
respectively. Dashes denote gaps.
Architecture of the Two BTB
Proteins in Ostreococcus lucimarinus
37 known yeast, plant, and animal BTB domains used as initial queries
BLASTp searches of the Chlamydomonas reinhardtii v5.3.1 predicted proteome and the
Ostreococcus lucimarinus v. 2 predicted proteome were conducted in the DOE-JGI comparative
plant genomics portal Phytozome (v9.1 [Chlamydomonas], v10.1 [Ostreococcus])
Species
Gene #
52 loci encoding BTB domain-containing proteins identified in Chlamydomonas and 2 loci
encoding BTB domain-containing proteins identified in Ostreococcus
4529
The predicted BTB domains from the proteins encoded by these loci were used in 2nd round
BLASTp searches of the Chlamydomonas and Ostreococcus predicted proteomes. Various
subsequent searches (BLASTp and tBLASTn) of the predicted proteomes and the whole genome
sequences were conducted to identify any genes missed.
27814
13 new loci encoding BTB domain proteins identified in Chlamydomonas. No new loci
encoding BTB proteins identified in Ostreococcus
MATH
ARM
Figure 4. Only two BTB genes were identified in
Ostreococcus. One is predicted to encode a protein with a
C-terminal MATH domain and the second is predicted to
encode a protein with Armadillo (ARM) repeats. MATHBTB and ARM-BTB proteins are also seen in
Chlamydomonas and land plants. MATH-BTB proteins are
also found in yeasts and in animals and are one of a very
few BTB types shared generally among the eukaryotes.
Final BTB families of 65 members (Chlamydomonas) and 2 members (Ostreococcus) identified
Phylogenetic Analysis of the BTB Families of
Chlamydomonas reinhardtii and Ostreococcus lucimarinus
BTB Gene Family Size and Genome Size
in Various Algal and Plant Species
Number of
BTB Family
Members
149(2)
Oryza sativa (rice)
Genome Size
372Mb
12 chromosomes
Arabidopsis
thaliana
81(1,2,3)
135Mb
5 chromosomes
Physcomitrella
patens
65(unpublished data)
473Mb
27 chromosomes
Chlamydomonas
reinhardtii
65
111Mb
17 chromosomes
Ostreococcus
lucimarinus
2
13.2Mb
21 chromosomes
0
0
1
6
0
0.2
3
3
5
Conclusions
0
0
73
29
17
16
39
27
23
1
20
2
1
3
37
27
32
80
83
90
56
Cre02.g096700
Cre10.g440800
BACK
Cre06.g286700
Cre03.g165801
PHR
Cre11.g480050 MATH
33
Cre11.g479800 MATH
Cre06.g278184 MATH
63
37
Cre01.g022950 MATH
Cre01.g048500 MATH
Cre07.g335950 MATH
Cre03.g151050 MATH-Like
Cre17.g700550 MATH-Like
97
MATH-Like
78 Cre17.g700600
Cre03.g174200
Cre14.g618900
Cre12.g520900
Cre04.g221900
Cre02.g113809
Cre17.g731466
Cre13.g583150
99 Cre14.g614500
Cre09.g388652
NHL
SBP/BACK
Cre03.g174150
Cre02.g101150
Cre03.g173165
NHL
Cre04.g215450
Cre04.g213650
Cre04.g220250
32
Cre02.g101550
14
Cre09g.401219
Cre12.g532900
Cre04.g219600
Cre04.g219400
Cre04.g212100
Cre02.g101300
5
6
69 Cre14.g610550
90 Cre14.g610582
Cre14.g610631
Cre04.g218400
Cre03.g179650
Cre16.g653258
Cre12.g542650
96
Cre10.g464450 BACK
BACK
SBP
Cre13.g585250
Cre10.g419850
MATH
ARM
ARM
Cre03.g205400
Cre13.g580450
Cre07.g342000
Cre03.g144284
Cre04.g231516
27814
SBP
Cre01.g012200
SBP
SBP
Cre17.g698233
Cre09.g399289
Cre16.g680750
Cre16.g680800
MATH
80
Cre05.g245101
4529
Cre01.g001600
90
Cre02.g110150
Cre04.g211700
7 67
4
27
18
56
67
79
85
25
82
56
19
34
75
90
Cre03.g167850
Cre16.g679876
Cre04.g221750
Cre04.g221770
Cre11.g467776
Cre03.g162200
= Ostreococcus
BTB family
member; the
remainder are
Chlamydomonas
BTB family
members
Figure 2. Phylogenetic tree of BTB family members from Chlamydomonas reinhardtii and Ostreococcus lucimarinus. The tree was generated in MEGA
6.06 by maximum-likelihood analysis of a Gblocks-edited alignment of the amino acid sequences of BTB domains from the family members. The tree
with the highest log likelihood is shown. Bootstrapping with 500 replicates was performed; the numbers at each node indicate bootstrap values.
Bootstrap values >75% indicate moderate to strong support for that node. Branch lengths are scaled to number of substitutions per site. Other
domains or motifs identified by SMART and/or Pfam in the predicted proteins are indicated for some family members.
•The BTB family in Chlamydomonas reinhardtii is large (65 members) and complex, but the family in
Ostreococcus lucimarinus is very small (2 members).
•The two Ostreococcus BTB genes appear to each have a homolog in Chlamydomonas (27814 and
Cre04.g231516; 4529 and Cre03.g144284).
•While the architectures found in the two Ostreococcus BTB proteins are also found in BTB proteins
from land plants and Chlamydomonas (MATH-BTB and ARM-BTB), most of the architectures found
in Chlamydomonas (e.g. BTB-SBP, NHL-BTB) are not seen in land plants and vice-versa.
•There is significant sequence diversity within the collection of Chlamydomonas BTB domains. This
is similar to the BTB domains found in animals and plants, which are quite variable mostly yet share
a common function (Cullin 3 protein interaction).
•Increasing genome size is generally associated with increasing BTB family size.
•These results suggest there have been drastic changes in the size and composition of this E3
ubiquitin-ligase gene family during Viridiplantae evolution.
References
1. Gingerich, D.J., 1, Gagne J.M., Salter, D.W., Hellmann, H., Estelle, M., Ma, L., Vierstra R.D. (2005) Cullins 3a and 3b Assemble with Members of the Broad Complex/Tramtrack/Bric-a-Brac (BTB) Protein Family to Form Essential Ubiquitin-Protein Ligases (E3s) in
Arabidopsis. Journal of Biological Chemistry, Vol. 280, No. 19, pp. 18810 - 18821
2. Gingerich, D.J., Hanada, K., Shiu, S-H, and Vierstra, R.D. (2007) Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/ Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice. The Plant Cell. Vol. 19, No. 8 , pp. 2329-2348
3. Christians, M. J.; Gingerich, D. J.; Hua, Z.; Lauer, T.D.; Vierstra, R. D. (2012). The Light-Response BTB 1 and 2 Proteins Assemble Nuclear Ubiquitin Ligases That Modify Phytochrome B and D signaling in Arabidopsis. Plant Physiology. 160(1), 118-134
Acknowledgements
This research is supported by University of Wisconsin-Eau Claire Differential Tuition
through the UWEC Office of Research and Sponsored Programs. We thank UWEC’s
Learning and Technology Services for the printing of this poster.