Extensins
Rich Wiemels
Assembly of cell wall proteins
•Proteins synthesized and hydroxylated
in the ER
•Glyosylation occurs in the Golgi
•Golgi vesicles secrete monomers to the
wall
•Monomers polymerize
•Cross-links form
•Ionic, covalent, H-bond,
electrostatic etc.
•Elicitor stimulated cross-linking
•How does self assembly of the cell wall
occur at the cell plate during cell
division?
Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants
Plant Cell Wall Structural Proteins
• Characteristics:
– Sequence information
• Motifs, palindromes, predictions
– Physical properties
• Solubility, charge, structure, etc.
– Post-translational modifications
• Hydroxylation (HRGPs)
• Glycosylation (amount, sugars involved, branching)
• Intramolecular cross-linking
– Interaction with other molecules
• Ionic interactions, salt bridges
• Intermolecular interactions
HRGPs
• 3 types
– Proline-rich proteins
• Lowly glycosylated, highly periodic
• Ara and Gal
– Extensin
• Moderately glycosylated, less periodic
• Ara and Gal
– Arabinogalactan proteins
• Highly glycosylated, least periodic
• Ara, Gal, Fuc, Rha, GlcNAc
Extensin Outline
•
•
•
•
Phylogeny
Motif comparisons
Purification
Cross-linking
– Intramolecular
• IDT
– Intermolecular
Ara on Hyp (contiguous only)
Gal on Ser
• Peroxidase, elicitors
– di-IDT, pulcherosine
• Extensin pectate
• Formation of extensin scaffold (today’s paper)
Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants
Overall phylogeny of HRGPs
Kieliszewski and Lamport 1994
Motifs
P hydroxylated to
Hyp (O)
PYYPPH and
TPVYK
Memelink et al. 1993
The most common extensin motif is Ser-(Hyp)4
Hydrophobic regions span intervals, very insoluble
VYK –putative intermolecular crosslinking site (Schnabelrauch et al. 1996)
Comparing Motifs
Kieliszewski and Lamport 1994
P1, P2 and P3 designated to differing extensin motifs
Note YKYK in tomato P2, isodityrosine (IDT) motif (Tyr-X-Tyr-Lys)
P1, P2, and P3
• P1: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys
– No IDT motif
• P2: Ser-Hyp-Hyp-Hyp-Hyp-Val-Tyr-Lys-Tyr-Lys
• P3: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-HypHyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys
– More Tyr cross-linking possibilities
Smith et al. 1986
Purifying Extensin
• Monomers soluble until incorporated into wall,
how to purify?
• Solubilize Hyp (Qi et al. 1995)
–
–
–
–
–
Digest homogalacturonan (EPG)
Digest cellulose, XyG (Cellulase)
HF at -73° to cleave furanosyl linkages, Ara removed
HF at 0° to cleave off all sugars
Ammonium carbonate to remove ionic interacting
molecules
Purifying extensin, pectin cross-link
Digests homogalacturonan
Remove cellulose and XyG
Remove furanosyl linkages (Ara)
RG-I still present
Remove ionically
associated
polymers
Remove all sugars
RG-I remains in an insoluble fraction, suggesting covalent linkage to pectin
Cross-links
• Peroxidase cross-linking
– Needed for deposition, cross-links Tyr
– Elicited defense mechanism
• IDT formation
– Added insolubility and strength
– Tyrosine tetramer (di-IDT) and trimer
(pulcherosine)
• Extensin pectate
– New model for wall assembly
Extensin Peroxidase (EP)
• Polymerizes extensin monomers (Schnabelrauch
1996)
• Oxidative cross-linking occurs at perception of
stress or elicitors (Bradley et al., 1992)
– Protects plant from pathogens, invaders
• Cross-linking occurs before transcription
dependent response (Brisson et al. 1994) and
after (Showalter, 1993).
• VYK possible cross-linking motif for EP in addition
to Tyr motifs (Schnabelrauch et al., 1996)
Various elicitors tested
for cross-linking
stimulation of GvP1, an
extensin from
grapevine
Jackson et al. 2001
Elicitors cause cross-linking and increased insolubility of extensin
Tyrosine linkages
• Tyr-X-Tyr-Lys motif
Isodityrosine (IDT) intramolecular linkages stabilizes extensin (P2, P3) and does
not disrupt helical conformation (Epstein and Lamport, 1984)
di-IDT and pulcherosine:
Intermolecular cross-linking
Brady and Fry, 1998
di-IDT and pulcherosine:
synthetic gene vs. purified extensin
• di-IDT (tetromer) forms in vitro from synthetic P3
extensin (Held et al., 2004)
– SPPPPYYYKSPPPPSP repeated 20x = (YK)20
• Very little di-IDT observed in RSH, an Arabidopsis
extensin. Instead Tyr trimer, pulcherosine (Cannon et
al. 2008)
?
…and that brings us to today’s paper
Self-assembly of the plant cell
wall requires an extensin scaffold
Maura C. Cannon1, Kimberly Terneus2, Qi Hall1, Li Tan2,
Yumei Wang1, Benjamin L. Wegenhart2, Liwei Chen2, Derek
T. A. Lamport3, Yuning Chen2, and Marcia J. Kieliszewski2
1. Department of Biochemistry and Molecular Biology, University of Massachusetts
2. Department of Chemistry and Biochemistry, Ohio University
3. Department of Biology and Environmental Science, University of Sussex, UK
The RSH mutant
• -root, -shoot, -hypocotyl
defective
• Identified by Hall and
Cannon 2002
• Major developmental
problems
– Severely misshapen cells
– Misplacement of cell plate
Extensins in Arabidopsis
• 20 homologous genes, but this one knockout has lethal phenotype!
rsh/rsh phenotype
Heart stage
embryos
Root sections
C-F rsh/rsh phenotype includes incomplete (floating, hanging) walls and wall stubs
Purifying and Identifying RSH
• Lys-rich positively charged extensin monomers salt eluted
– Superose-6 gel filtration yielded monomer
– Cation-exchange chromatography and alkaline hydrolysis yielded
extensin arabinooligosaccharides
• Deglycosylation by HF and sequencing confirmed identity as AtEXT3
Protein Sequence
Highly periodic. 11 identical 28-residue repeats, monomer = 308 amino acids
Cross-linking Assay
•Pulcherosine is more prevalent than di-Idt as the
cross-linking tyrosine derivative
Calculating Types of Tyr derivatives
•Very little di-Idt forms unlike (YK)20 (Held et al. 2004)
•Why are pulcherosine and Idt the dominant Tyr cross-linking derivatives?
Explaining di-Idt absence
•Parallel alignment, as with (YK)20 yields only di-Idt motifs (A)
•Staggering the alignment yields only pulcherosine motifs– Ser-(Hyp)4 still aligned (B)
C-terminal significance
• GFP fused to C-terminus did not rescue rsh/rsh
double mutant
• N-terminal fused GFP yielded functional RSH
AFM data
(YK)20
RSH monomer
-Forms dendritic
structure
•Segments calculated to be 127nm for polyproline II helical secondary structure
•Molecules are overlapping, stretch longer than RSH molecule from staggered
alignment
Self Assembly
• Monomers of extensin self assemble into
dendritic scaffold
– Consistent with other self-assembling amphiphiles
at liquid interfaces (Rapaport 2006)
– Able to form template for pectin
• New paradigm for cell wall assembly
– Extensin needed at cell initiation, not cessation of
growth
Conclusions: A new paradigm for cell
wall self-assembly
1. Liquid-liquid interface promotes self-assembly of
ordered amphiphilic arrays
2. Alternating hydrophilic/phobic modules induce selfrecognition
3. Periodicity aligns monomers
4. C-terminus sequence may initiate end-on adhesion
5. Intermolecular Tyr cross-links stabilize
6. Pulcherosine cross-links favor staggered RSH alignment
7. Staggered alignment allows 2D growth
8. Extensin and pectin form extensin pectate
9.Extensin scaffolds template for orderly pectic matrix