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The Pennsylvania State University
The Graduate School
Biochemistry, Microbiology, and Molecular Biology Department
COMPLEX FORMATION AND PROTEIN INTERACTION IN THE CELLULOSE
SYNTHESIS COMPLEX OF ARABIDOPSIS
A Thesis in
Biochemistry, Microbiology, and Molecular Biology
by
Ashley Nicole Hill
 2016 Ashley Nicole Hill
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2016
ii
The thesis of Ashley Hill was reviewed and approved* by the following:
Ming Tien
Professor of Biochemistry
Thesis Advisor
Ying Gu
Assistant Professor of Biochemistry and Molecular Biology
Sarah Ades
Associate Professor of Biochemistry and Molecular Biology
Tracy Nixon
Professor of Biochemistry and Molecular Biology
Scott Selleck
Professor of Biochemistry and Molecular Biology
Head of the Department of Biochemistry and Molecular Biology
*Signatures are on file in the Graduate School
iii
ABSTRACT
All plant cells are encapsulated by cell walls, which serve multiple roles in plant growth,
strength, and protection. This plant wall material is manufactured incessantly and although the
composition may vary as a function of location, developmental stage, and environment, this material
is composed of polysaccharides, proteins, and (in secondary cell walls) lignins (Somerville et al.
2004). Despite several decades of research and recent increased funding due to the economic pushes
for advancements in the field of renewable energy, still little is known about the biochemistry of the
biosynthesis of one of these polysaccharides: the β-(1,4)-linked glucan, cellulose. This work’s focus
was on exploring possible methods through which the cellulose synthesis proteins of Arabidopsis
thaliana may interact to form the cellulose synthesis complex. Work focused on the N-terminal
domains of the cellulose synthase proteins showed extensive homo-oligomerization that could be
altered through Cys-->Ala mutations in the zinc-finger domain.
Work focused on in vivo
complementation of mutant Arabidopsis plants with domain-swapped CesA proteins yielded plant
transformants that may be screened for phenotypic complementation and assayed for cellulose
content.
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TABLE OF CONTENTS
List of Figures .......................................................................................................................... v
List of Tables ........................................................................................................................... vi
Chapter 1 The Current Known Composition and Interactions of the Cellulose Synthesis
Complex ........................................................................................................................... 1
Cellulose Synthase Complex Composition ...................................................................... 1
CesA and Cellulose Biosynthesis in Arabidopsis ............................................................ 2
CesA-CesA Interaction .................................................................................................... 5
Chapter 2 Characterization of CesA Interaction through the N-terminal Domain .................. 7
Introduction ...................................................................................................................... 7
Results .............................................................................................................................. 8
Discussion ........................................................................................................................ 15
Materials and Methods ..................................................................................................... 16
Chapter 3 Assessing CesA-CesA interactions through in vivo Domain Swaps ....................... 17
Introduction ...................................................................................................................... 17
Results and Discussion..................................................................................................... 18
Materials and Methods ..................................................................................................... 22
References ................................................................................................................................ 24
v
LIST OF FIGURES
Figure 1. CesA Topology
3
Figure 2. CSC Models
6
Figure 3. N-termini oligomerization on SDS PAGE
8
Figure 4. Anaerobic N-termini oligomerization on SDS PAGE
9
Figure 5. Anaerobic N-termini oligomerization on Native PAGE
9
Figure 6. Effect of reducing and chelating agents on CesA N-termini oligomerization.
10
Figure 7. FPLC of CesA3 n-terminus
12
Figure 8. Redistribution of monomeric protein into a mixed population.
12
Figure 9. FPLC of dithionite-treated CesA3 n-Terminus
13
Figure 10. Triton X-100 treatment of CesA N-termini
13
Figure 11. Comparing CesA6 and CesA6 CA n-termini oligomeric distributions
14
Figure 12. Schematic of CesA 1/6 chimeric proteins.
19
Figure 13. Schematic of CesA 4/8 chimeric proteins.
19
Figure 14. Dwarf and nearly sterile phenotype of irregular xylem mutant.
21
vi
LIST OF TABLES
Table 1. DLS data from WT and mutant CesA6 N-termini. ...................................................................... 14
Table 2. Primers for N-terminus cloning ................................................................................................. 16
Table 3. Primers for chimeric CesA construction .................................................................................... 23
Chapter 1
The Current Known Composition and Interactions of the Cellulose Synthesis
Complex
Cellulose Synthase Complex Composition
One of the first major advances in understanding the biochemistry of cellulose
biosynthesis was the visualization cellulose microfibrils being extruded from protein globules,
called terminal complexes, in cells of higher plants through freeze-fracture electron microscopy
(Mueller et al. 1976). These terminal complexes were later identified as forming a hexameric
“rosette” structure in the plasma membrane and named the Cellulose Synthesis Complex (CSC)
(Mueller & Brown 1980; Desprez et al. 2007). It then took nearly two decades to identify the
main components of the CSC as a family of glycosyltransferase proteins known as cellulose
synthases (CesAs) (Kimura et al. 1999). In Arabidopsis, there are ten similar, yet distinct, CesA
isoforms (Richmond & Somerville 2000). CesA genes were originally identified, in early cDNA
sequence data, by their homology to the cloned and sequenced cellulose synthase from the
bacterium Gluconacetobacter xylinum (Pear et al. 1996).
Based on measurements of microfibrils in electron microscopy images, and the 25-30 nm
diameter of the rosette in freeze-fracture electron microscopy, it was estimated that the cellulose
microfibril contains 36 cellulose chains, and as a result, it was assumed that the CSC contains 36
CesA proteins (Mueller & Brown 1980; Delmer 1999; Herth 1983b). This roundabout estimation
for the number of CesAs within the CSC has persisted for decades, but is currently being called
into question by more recent and accurate measures that are more consistent with an 18-24 glucan
2
chain cellulose microfibril (Ha et al. 1998; Buleon & Chanzy 1978; Endler & Persson 2011;
Thomas et al. 2013; Fernandes et al. 2011).
Besides the CesA proteins themselves and KORRIGAN, a β-1,4 glucanase (Vain et al.
2014), there has not been sufficient evidence of other proteins as integral components of the
CSC. Several proteins have been shown to interact with the CesAs and the CSC, however
sometimes this interaction is transient. These interacting proteins include cellulose synthase
interacting protein 1 (CSI1), a microtubule interacting protein, and, more recently, AP2M and
TWD40-2, both part of the clathrin-mediated endocytosis machinery (Gu et al. 2010; Li et al.
2012; Bashline et al. 2013; Bashline et al. 2015; Vain et al. 2014). In addition to the known
interacting proteins, genetic and biochemical evidence has implicated several other proteins that
are hypothesized to be interacting or associated with the CSC.
The GPI-anchored protein
COBRA is thought to have a role in microfibril orientation and crystallization (Schindelman et
al. 2001; Liu et al. 2013; Sorek et al. 2014). Mutants in the plasma membrane-localized protein of
unknown function KOBITO, display defects in cellulose biosynthesis (Pagant et al. 2002).
Sucrose synthase is thought to interact with the CSC to rapidly supply the substrate, UDPglucose, via substrate channeling (Coleman et al. 2009; Amor et al. 1995).
CesA and Cellulose Biosynthesis in Arabidopsis
Although the structure of a bacterial cellulose synthase has recently been solved (Morgan
et al. 2013) we possess limited structural understanding of plant CesAs. Additionally, many
biochemical parameters remain elusive. These include the catalytic mechanism, the entire suite of
proteins within the CSC, organization and protein interactions between CesAs, and the regulatory
system of cellulose biosynthesis, including the timing and purpose of phosphorylation (Chen et
al. 2010; Guerriero et al. 2010; Li et al. 2014). Of the ten Arabidopsis
CesA
isoforms
3
(Richmond & Somerville 2000), i) three (CesA1, 3, 6) interact to form a CSC responsible for
cellulose biosynthesis in the primary cell wall, which is the ‘strong but pliable’ wall present
during cell division and growth (Persson et al. 2007; Cosgrove 2005), ii) three other CesAs
(CesA4, 7, 8) form a separate and distinct CSC for cellulose biosynthesis in the secondary cell
wall, which is a thick and rigid, lignified wall deposited in select cells after cessation of growth
(Taylor et al. 2003; Cosgrove & Jarvis 2012; Cosgrove 2005), iii) and the remaining four CesA
isoforms, CesAs 2, 5, 9, and 10, are involved in various tissue specific processes, including
gamete formation, flowering, and biosynthesis of seed coat mucilage (Mendu et al. 2011; Sullivan
et al. 2011; Stork et al. 2010; Harpaz-Saad et al. 2012; Persson et al. 2007). Additionally, CesAs
2 and 5 have been shown to be partially redundant with CesA6 (Desprez et al. 2007; Persson et al.
2007).
Figure 1. Predicted topology of CesAs, depicting the central domain (containing the
D, D, D, QxxRW catalytic motif and the class-specific region [CSR]), the N-terminus
(containing the zinc finger domain [Zn] and the hyper variable region [HVR]), and
the C-terminus localized to the cytosol.
As in all higher plants, the 10 CesA isoforms of Arabidopsis are highly homologous,
containing only two clusters of significant sequence divergence (Yin et al. 2009; Carroll &
Specht 2011; Richmond & Somerville 2000). One region is referred to as the hyper variable
region (HVR) and is located in the purportedly soluble N-terminal domain and shows extremely
high levels of divergence between CesAs. The other is referred to as the class specific region
(CSR), located in the central “catalytic” domain, which possesses divergence between CesA
4
paralogs, but homology between orthologs (Vergara & Carpita 2001). Other features of possible
importance, illustrated in Figure 1, are a cysteine-rich RING-type zinc finger domain located in
the N-terminus, the D, D, D, QxxRW highly-conserved catalytic motif characteristic of
processive glycosyltransferases, and the plant conserved region (PCR) that is completely absent
from the bacterial cellulose synthase but is highly conserved among plant CesAs (Sethaphong et
al. 2013; Pear et al. 1996). Although it is most logical for the catalytic domain to face the cytosol,
where it would be most accessible to the substrate, the topological model of the protein in Figure
1 has yet to be demonstrated empirically.
Biochemically, we know the CesA protein is responsible for catalyzing the addition of a
glucose residue from the activated substrate UDP-glucose to the growing cellulose chain, forming
a β-1,4 glycosidic bond (Morgan et al. 2013). While, the mechanism by which this occurs
remains elusive, several mechanisms of other glycosyltransferases have been determined
(Lairson et al. 2008; Amor et al. 1995). Unfortunately, plant CesAs have resisted traditional
biochemical characterization, often losing all cellulose synthesis activity upon attempts to purify
(Brown Jr. et al. 1994). However, some in vitro cellulose synthesis activity has been achieved
from crude membrane preparations of plant suspension cell cultures, though the major glucan
synthesis activity in these systems is for the β-1,3 glucan callose (Lai-Kee-Him et al. 2001; LaiKee-Him et al. 2002; Colombani et al. 2004; Cifuentes et al. 2010). To extrude the cellulose
polymer, it is proposed that the transmembrane domains of CesA proteins themselves form a pore
or a barrel through which the elongated chain is extruded from the cytoplasmic membrane and
deposited into the plant cell wall (Buckeridge et al. 2001; Buckeridge et al. 1999).
5
CesA-CesA Interaction
The unique rosette structure of the CSC, in contrast to linear arrays of synthases observed
in bacteria, is thought to play a significant role in the final crystal structure of the cellulose
microfibril (Kimura et al. 2001; Sunagawa et al. 2013). Indeed, alterations in the rosette structure
correlate to difference in cellulose structure (Brown Jr. & Montezinos 1976; Herth 1983a). Thus,
it is of great importance to understand the structure and assembly of CesAs into the CSC.
Although the number of CesAs within the CSC has not been empirically determined, it has
traditionally been estimated at 36 CesAs, though there has been a recent shift in support of an 18CesA CSC (Doblin et al. 2002; Hill et al. 2014; Newman et al. 2013). Recently, it has been
determined that CesA isoforms exist in a 1:1:1 stoichiometry (Hill et al. 2014). However the
organization and interaction between CesAs within the CSC has yet to be determined
unambiguously. There have been several somewhat inconclusive and contradictory studies
attempting to address the question of CesA-CesA interaction (Carroll et al. 2012; Li et al. 2013;
Timmers et al. 2009; Desprez et al. 2007). It has been shown through yeast two-hybrid (Y2H)
work that the N-terminus region may be responsible for mediating the interaction between CesA
proteins (Timmers et al. 2009). A 3:2:1 stoichiometric model (Figure 2A) has been generated
based on those Y2H results along with bimolecular florescence evidence from the same group. It
was proposed that the zinc-finger region within the N-terminus may be key for this interaction;
however mutations of several cysteine residues decreased, but were unable to abolish the
interaction between secondary cell wall CesA proteins (Timmers et al. 2009).
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Figure 2. A-B) Speculative stoichiometric model of CSC (Timmers et al. 2009). C)
1;1:1 stoichiometric model based on quantitative Western blotting (Hill et al. 2014).
Work investigating the mechanism of cotton CesA subunit interaction in vitro has
determined that the cysteines within the N terminus region possibly form disulfide bonds, and
that the oxidation state of the protein regulates this interaction (Kurek et al. 2002; Atanassov et al.
2009). In a separate study, targeted domain swaps were used to produce chimeric proteins with
N-terminal regions “swapped” between CesAs 1 and 3 (Wang et al. 2006). Rescue of CesA 1 and
CesA3 swap mutants was correlated to the presence of the ‘correct’ C-terminal domain (catalytic
domain + transmembrane regions). Conversely, the reciprocal chimeras were unable to rescue the
mutants (i.e. a chimera with the CesA1 N-terminus could not rescue the cesa1 mutant) (Wang et
al. 2006). These results are consistent with the N-terminus as not mediating specific CesA-CesA
interactions. As mentioned previously, the current body of literature exploring CesA subunit
interaction is frequently in conflict and leaving us unable to derive concrete conclusions
regarding CesA-CesA interactions. The work described herein aims to expand our understanding
of how CesAs interact to form the CSC.
7
Chapter 2
Characterization of CesA Interaction through the N-terminal Domain
Introduction
Understanding the how CesA proteins interact is of great importance to the development
of a robust model of plant cellulose biosynthesis. In order to characterize the interaction between
the CesA proteins by quantitative means, we have studied the in vitro interactions between
Arabidopsis CesA N-terminal domains. Previously, heterologously-expressed cotton N-terminal
domains have been shown to dimerize, in a redox dependent manner, through their zinc binding
domains (Kurek et al. 2002). However, assembly of a functional CSC requires interaction
between three unique CesA isoforms, and Kurek and colleague’s previous study was only able to
investigate two CesA isoformss, as they were limited by the poorly characterized nature of the
cotton CSC system (Persson et al. 2007; Taylor et al. 2003; Kurek et al. 2002). Our current
analysis of interactions between Arabidopsis CesA N-termini takes advantage of the larger body
of knowledge that exists for Arabidopsis CSC composition. In Arabidopsis, it is known that
CesAs 1, 3, and 6 form a CSC during primary cell wall synthesis, while CesAs 4, 7, and 8 form a
separate and distinct CSC during secondary cell wall development (Taylor et al. 2003; Desprez et
al. 2007).
Using Arabidopsis CesA N-termini, this study attempts a more comprehensive analysis of
interactions between all CesAs within a CSC, utilizing techniques such as analytical
ultracentrifugation, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS).
Additionally, site-directed mutagenesis was used to identify residues important for these proteinprotein interactions. Additionally, we planned to quantify changes in interaction strength,
resulting from these amino acid substitutions, through monitoring the Kd between the proteins.
8
However, technical difficulties in working with the Arabidopsis N-terminal truncations limited
the final scope of our analysis.
Results
Five of the six most important Arabidopsis CesAs were expressed in E. coli with a
hexahistidine tag at the N-terminus. Each consists of the first ~180-280 amino acids of the
polypeptide.
Expression
of
these
truncated
domains was robust and purification to near
homogeneity was easily achieved by the Ni-NTA
affinity column. However, upon analysis by SDSPAGE and Western blotting, extensive homooligomerization of these proteins was observed,
even under the denaturing and reducing conditions
of the gel. As shown in Figure 3, each CesA Nterminus truncation exists in multiple populations.
The lane containing the
CesA1NT preparation
exhibited bands at ~34 and ~65kDa (consistent
Figure 3. Western blot (anti-His) from
SDS-PAGE gel showing oligomerization
of purified CesA N-termini.
with monomer and dimer populations). The CesA3NT preparation exhibited bands at ~35 and
~105kDa (monomer and trimer populations). The CesA6NT preparation is present as a 39kDa
monomer and also has a trimer band. CesA4NT is visualized as a 25kDa monomer and also
displays a trimer band. CesA8NT is detected as a 25kDa monomer and also as dimer and trimer
bands.
This formation of SDS, reducing agent-stable oligomerization was problematic, as my
research plan involved assessing the interactions between mixtures of different CesA’s N-
9
terminal domains.
This required monomer
starting materials. Previous work with cotton
CesA N-termini also formed oligomers, but these
could be readily dissociated to a monomeric state
with only 5 mM of the reducing agent
dithiothreitol (Kurek et al. 2002). This is just onetenth of the DTT concentration used in the blot
shown in Figure 3. Treatment of the samples with
an
even
stronger
reducing
agent,
tris(2-
carboxyethyl)phosphine (TCEP), did not result in
shifting the protein population toward the
Figure 4. Western blot from SDS-PAGE
showing oligomerization of anaerobically
purified CesA N-termini.
monomer as assessed by SDS-PAGE and
Western blotting (data not shown). In a further attempt to promote a reducing environment to
result in monomeric protein, I conducted cell lysis, protein purification, and SDS-PAGE analysis
under anaerobic conditions in a glove box. This anaerobic methodology reduced, but was not able
to abolish, homo-oligomerization on SDS-PAGE.
As shown in Figure 4, CesA1, 3, and 6 N-termini,
prepared anaerobically, still resulted in formation
of dimers (CesA1NT dimer band visible by eye but
contrast upon digitization is insufficient).
While anaerobic preparation resulted in a
high proportion of monomeric CesA-NT when
denatured and reduced during SDS-PAGE, it does
not reveal the oligomeric state of these proteins
Figure 5. Native-PAGE analysis of
anaerobically purified CesA N-terminal
domains. 1x, 2x, 4x, represents
proportional amounts of protein loaded.
10
under native (non-denaturing) conditions. So, to assess for their native oligomeric state, I
performed native-PAGE on these samples. This analysis revealed that a significant degree of
homo-oligomerization was still present (Figure 5). While anaerobic purification improved the
yield of monomeric CesA N-termini, the monomeric form was not sufficiently enriched for
analysis by isothermal titration calorimetry (ITC) or analytical ultracentrifugation, nor was it
monodispersed enough for crystallization.
Further treatment was necessary to obtain a
monodispersed sample. We hypothesized that treatment of anaerobically purified protein with
reducing agents would improve the chances of obtaining the necessary predominantly monomeric
population. We also wanted to explore the effect of chelating agents on oligomerization, due to
the possibility that zinc or other metal ions may have a role in mediating interactions between the
zinc-finger domains present in the CesA N-termini.
Samples were purified anaerobically and treated with 10mM DTT or 10mM DTT plus
1mM EDTA for one hour before separation on native-PAGE and visualization by Western blot
with anti-His antibody. Unfortunately, treatment with 10mM DTT decreased but failed to abolish
homo-oligomerization. Furthermore, as shown in Figure 6, treatment with EDTA had no obvious
effect on the sample’s oligomeric state.
Figure 6. Effect of reducing and
chelating agents on CesA N-termini
oligomerization. Native-PAGE and
western blot visualization
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
CesA1
CesA1 w/ 10mM DTT
CesA1 w/ 10mM DTT, 1mM EDTA
CesA6
CesA6 w/ 10mM DTT
CesA6 w/ 10mM DTT, 1mM EDTA
Ladder
CesA4
CesA4 10mM DTT
CesA4 10mM DTT, 1mM EDTA
11
While the combination of anaerobic purification and reducing agent did not result in
formation of 100% monomeric CesA N-termini, native-PAGE results suggested the majority of
protein was indeed monomeric (highest mobility bands in Figures 5 and 6). However, nativePAGE has limited interpretability, as there is no reliable molecular weight marker to
unequivocally determine if these high mobility bands were the monomer. In order to attain a more
reliable measure of molecular weight and to hopefully isolate a population of monomers, I turned
to size exclusion chromatography. Anaerobically purified and reduced CesA N-terminus protein
was separated by Fast Protein Liquid Chromatography (FPLC) on a Sephacryl S-300 column. I
obtained peaks corresponding to 80 and 25kD for the CesA3 n-terminus (a 29kD protein),
roughly the size of a monomer and a trimer (Figure 7). The 25kD fraction was collected and
analyzed on native-PAGE and Western blotting. The results revealed that, sometime during
manipulation and gel separation, the monomer fraction redistributes into monomer and oligomer
populations (Figure 8).
12
Figure 7. Chromatogram (OD280) of CesA3 n-terminus (29kD) FPLC separation
using S-300 column. X-axis = mL of elution
In an attempt to improve the FPLC separation, I included
the reducing agent dithionite to observe how it affected the
oligomeric distribution of the CesA3 n-terminus during
size exclusion chromatography. Purified CesA3 Nterminus protein was incubated with 50 mM dithionite for
one hour and then applied onto an S-300 column
equilibrated with a running buffer, also containing 50 mM
dithionite. I observed peaks corresponding protein at 50
and 25 kDa (Figure 9), indicating that the trimer
population had been reduced into dimers and possibly
monomers by the treatment.
Figure 8. Western blot from
native-PAGE using the 25kD
fraction from Figure 7 shows
redistribution of monomeric
protein into a mixed population.
13
Figure 9. Chromatogram (OD280) of dithionite-treated CesA3 n-Terminus separated on a S300 column equilibrated with 50mM dithionite running buffer. X-axis = mL of elution
In
another
attempt
to
obtain a monodispersed sample
without
using
treatments
that
would interfere with subsequent
biochemical analysis, I assessed the
ability for the gentle, non-ionic
detergent Triton X-100 to prevent
oligomerization.
Purified CesA1
Figure 10.. Western blot of denaturing PAGE gel
containing 0.2mM Triton X-100 of samples incubated
at a final concentration of 0.2mM Triton X-100. 1x,
2.5x, and 5x indicate relative loading amounts.
and 3 N-termini were incubated with 0.2 mM Triton X-100 for 30 min and then separated by
PAGE with gels also containing 0.2 mM Triton X-100. The results of this treatment are shown in
the Western blot of Figure 10. Triton X-100 treatment did not appear to aid in the isolation of
monomeric N-termini, as high-molecular weight oligomers are still observed.
14
Based on the large number of cysteines in the CesA Ntermini and previous work implicating the redox state of these
proteins in controlling their oligomerization (Kurek et al. 2002), I
hypothesized that the extensive oligomerization of my N-termini
proteins was via disulfide bond formation. So, in a final attempt to
obtain a sample of CesA N-terminus monomers for further
analysis, I purchased a synthetic CesA6 N-terminus gene where
each cysteine residue was substituted with an alanine, in order to
completely prevent disulfide formation. This CA mutant CesA6
N-terminus protein expressed and purified as well as the wild type
version. However, Western blot analysis of native-PAGE revealed
Figure 11. Western blot
of
Native
PAGE
comparing CesA6 and
CesA6 CA n-termini
oligomeric distributions.
that the CA protein still existed in multiple oligomeric forms
(Figure 11). Dynamic light scattering data indicated that the CA substitutions decrease the
level of oligomerization, with the wild type truncation existing as mostly a pentamer and the
majority of the mutant protein as a dimer. However, the CA mutant is still not monodispersed
and a significant percentage of the population remained a higher-order oligomer (Table 1).
Crystallization trials of the CesA6 CA mutant N-terminus protein failed to yield protein
crystals.
Table 1. DLS data from WT and mutant CesA6 N-termini.
This shows that mutation of the cysteines to alanine alters oligomeric distribution, but does
not result in a fully monomeric, monodispersed sample.
15
Discussion
The goal of my research was to heterologously express, purify the N terminal domains of
different CesAs to characterize their interactions.
The rationale for this research was to
determine, through these in vitro studies, the nature of CesA assembly into the CSC. While the
Arabidopsis CesA N-termini truncations expressed at a high level in E. coli and were able to be
purified with a Ni-NTA affinity column, I was unable to obtain these proteins in a monomeric
form. This is a prerequisite for further experimentation. I hypothesized that the oligomerization
of these proteins was mediated by disulfide bonds between the many cysteine residues present
within the polypeptides. Various treatments, such as DTT, anaerobic purification, and dithionite
were able to shift the distribution of the CesA N-termini towards a monomeric state, but failed to
generate fully monomeric protein (Figures 4, 5, 6, 7, 8, and 9). While these results are consistent
with disulfide linkages between N-termini mediating some of the protein-protein interactions,
disulfide bonds cannot account for all of the observed oligomerization. This is best demonstrated
by the substitutions of every cysteine in the CesA6 N-terminus with alanine, resulting in a protein
incapable of disulfide bond formation. In the case of this CA mutant form of the CesA6 Nterminus, oligomerization was reduced but not completely eliminated, as assessed by nativePAGE and dynamic light scattering (Figure 10 and Table 1). These results suggest that a
significant amount of CesA N-terminus in vitro oligomerization occurs via a non-covalent
interaction, possibly hydrophobic. Additionally, the data presented here cannot distinguish
between specific and non-specific interactions, leaving the possibility that CesA N-terminal
domain truncations are simply very ‘sticky’. Unfortunately, monomeric CesA N-terminus protein
was never obtained so analysis of hetero-oligomerization could not be undertaken.
16
Materials and Methods
CesA N-termini were PCR amplified from cDNA using the primers outlined in Table 2.
PCR products were digested with NdeI and NotI, then ligated into NdeI/NotI cut pET21a, and
sequence verified. Constructs were introduced into BL21 (DE3) cells and protein expression was
induced by the addition of 1 mM IPTG. Protein purification over NiNTA was carried out
according to manufacturer’s instructions (QIAexpressionist, June 2003, Qiagen). SDS-PAGE was
conducted by the method of Laemelli (Laemmli 1970). Native-PAGE was carried out essentially
the same way, with the omission of SDS and denaturation by heating.
Table 2. Primers for N-terminus cloning
Start codon in all caps, restriction sites underlined
17
Chapter 3
Assessing CesA-CesA interactions through in vivo Domain Swaps
Introduction
Previously, the interaction between CesA proteins of the primary cell wall was
investigated by a series of domain swaps targeting the CesA N-terminus (Wang et al. 2006). For
this study, the N-termini of CesA1 and CesA3 were swapped and rescue of cesa1 and cesa3
missense mutants was attempted. However, the results from these chimeric proteins were
inconclusive. The results suggest that the N-terminus is likely not involved in specificity of
interaction between the CesA proteins (Wang et al. 2006). This is in contradiction to other
studies which indicate the CesA N-terminus plays a role in CesA-CesA interactions (Kurek et al.
2002; Timmers et al. 2009). Thus, it remains unclear how the plant or the cellulose synthesis
complex itself is differentiating between the various CesA isoforms present to correctly assemble
a functional CSC.
In addition to the N-terminus, there are two regions of interest that may be involved in
CesA-CesA interaction: the class specific region (CSR) and the C-terminus. The CSR is located
within the catalytic domain of the protein. It is highly conserved between CesA orthologs and
variable between CesA paralogs (Vergara & Carpita 2001). This is consistent with a conserved
function specific to each CesA ‘class’, such as mediating the specific CesA-CesA interactions
necessary for placement within a CSC. The C-terminus remains an area of interest mainly due to
inferences drawn from the crystal structure of an enzyme that shares some homology with
cellulose synthase, the chitin synthase SpsA of Bacillus subtilis. With this protein, presence of the
18
C-terminus is required for crystallization as a homo-dimer, while truncation to remove the Cterminus resulted in SpsA crystallizing as a monomer. (Charnock & Davies 1999). Thus, it is
feasible that any of these three regions of CesA (N-terminus, CSR, or C-terminus) are responsible
for recognition of isoform specificity in assembling the CSC. In order to comprehensively test
these three regions, this work aims for a more comprehensive chimeric CesA study than what has
previously been reported (Wang et al. 2006). I have constructed every permutation of swap
between these three domains between CesAs 1 and CesA6 and between CesAs 4 and CesA8.
Results and Discussion
Due to the intricacies of CSC formation and the possible contribution of intracellular
processing in determining protein interactions, it is important to carry out in vivo studies of CesA
interaction. Using the Geneart seamless cloning strategy (Invitrogen), I generated two series of
constructs consisting of 12 total chimeric CesA genes swapping three different CesA domains.
These chimeric genes were then to be assessed in vivo by their ability to rescue cesa mutants. I
took a conservative approach in designing these chimeras. The CesA were split into three broad
domains, delineated by transmembrane helix (TMH) regions, which have high levels of
homology between CesAs. These three regions consisted of the N-terminus + TMH 1 & 2
[labeled N], the central domain (containing the CSR) [labeled D], and TMH 3-6 + the C-terminus
[labeled C]. Figures 12 and 13 show a schematic representation of the 12 chimeric CesA genes
that I generated.
19
Figure 12. Schematic of CesA 1/6 chimeric proteins.
Figure 13. Schematic of CesA 4/8 chimeric proteins.
20
One major weakness of previous experiments with chimeric CesAs was difficulty
interpreting the results (Wang et al. 2006). This was primarily due to the weak cesa mutant alleles
that were used for the in vivo complementation assay. The CesA1 (rsw1-1) and CesA3 (rsw5)
alleles are each missense mutations with subtle mutant phenotypes, which only become
significant at elevated temperature (Wang et al. 2006). Additionally, the use of missense mutants
is problematic because the mutant CesA will be present alongside a chimeric transgene,
potentially confounding the results. To overcome this problem, this work uses cesa null mutants,
providing a cleaner genetic background. These mutants include: 1) a CesA1 T-DNA null insertion
we refer to as cesa1-2 that has no obvious phenotype when heterozygous, but gametophytic lethal
(Persson et al. 2007), 2) the temperature-sensitive CesA1 A549V substitution that results in radial
swelling of the root tips in plants grown at 31°C (rsw1-1) (Arioli et al. 1998), 3) a CesA6
nonsense mutation (Q720*) that presents as a severe growth defect in dark-grown hypocotyls
(prc1-1) (Hématy et al. 2007; Fagard et al. 2000), 4) a CesA8 T-DNA null insertion (irx1-5)
resulting in small, dark green plants with irregular xylem, and 5) a CesA4 T-DNA null insertion
(irx5-4) that results in small, dark green plants with irregular xylem.
21
The research strategy involved introducing the chimeric CesA genes, driven by a native
CesA promoter, into each respective cesa mutant line. I would then determine if they can
functionally rescue the mutant. I first focused on the CesA4/8 chimeras, as the secondary cell
wall irx mutants display a clear and
distinctive phenotype. Unfortunately,
although several attempts were made,
I failed to get antibiotic/herbicide
resistant Arabidopsis transformants.
This was likely due to the near sterility
of the irregular xylem mutants. As
illustrated in Figure 14, irx plants are
dwarf and frequently have difficulty
producing
productive
siliques.
Occasionally, irx plants will display
high levels of fecundity, but we have
yet to understand the basis of this. It is
possible this process is stochastic or a
certain set of growth conditions that
sometimes
occurs
to
facilitate
pollination and seed maturation.
Figure 14. Dwarf and nearly sterile phenotype of
irregular xylem mutant.
22
This project was planned to be multi-stage, with stage one being outlined above. One
subsequent stage was to involve generating further swaps, utilizing the same three domains, but
incorporating CesAs 3 and 7. I also planned to iterate on this domain swap process. This first
round used very large regions. But, if any chimera were to successfully rescue, I would be able to
use that information to subdivide one of the stage 1 domains in order to narrow down the region
responsible for specific CesA incorporation into the CSC. If I had been able to generate
transgenic plants, I believe at least some chimeric CesA genes would have been able to rescue.
The stage one constructs were designed very conservatively, choosing large regions and points of
high conservation for the splice sites, minimizing the likelihood of unintended protein misfolding. This methodology has already been shown feasible by the CesA 1/3 N-terminus chimeras
(Wang et al. 2006). Furthermore, a chimera where the central domain of cellulose synthase-like
D3 (CslD3) was replaced by the central domain of CesA6 was able to rescue the kjk-2 null CslD3
mutant (Park et al. 2011). In any case, I was able to successfully generate 12 chimeric CesA gene
constructs in plant transformation vectors, which may be of future use to the Tien lab.
Materials and Methods
Gene fragments corresponding to the N-terminal [N], Central [D], or C-terminal [C]
domains were amplified from CesA cDNA template, for each CesA, using the primers outlined in
Table 3. Chimeric CesA constructs were assembled in a Geneart™ seamless cloning reaction
(Invitrogen) by incubation of N, D, and C insert PCR fragments with NotI/PstI digested vector.
For CesA 1/6 chimera, the vector was pORE-O4 containing 2.5kb of the native CesA1 promoter
region (Coutu et al. 2007). For CesA 4/8 chimera, pORE-O3 containing 2.5kb of the native
CesA4 promoter region was used. Constructs were sequence verified, transformed into
Agrobacterium, and then these Agrobacterium were then screened to verify the presence of the
23
chimeric CesA construct. The CesA mutants rsw1-1 (CS6554), prc1-1 (CS297), irx5-4
(SALK_084627), irx1-5 (SALK_026812C), and cesa1-2 (CS812877) were obtained from the
Arabidopsis Biological Resource Center. Chimeric CesA constructs were transformed into these
mutant Arabidopsis by the floral dip method (Clough & Bent 1999). Seeds were selected in
solium, by spraying germinated seedlings with 75 ug/mL glufosinate ammonium (Sigma).
Table 3. Primers for chimeric CesA construction
Red = region binding to template DNA for PCR
Black = ‘tail’ to provide homology overlap region needed for seamless cloning reaction
24
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