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Extensins

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.

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Extensins

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  1. Extensins Rich Wiemels

  2. 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

  3. 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

  4. 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

  5. Extensin Outline • Phylogeny • Motif comparisons • Purification • Cross-linking • Intramolecular • IDT • Intermolecular • Peroxidase, elicitors • di-IDT, pulcherosine • Extensinpectate • Formation of extensin scaffold (today’s paper) Ara on Hyp (contiguous only) Gal on Ser Buchanan, Gruissem and Jones. (2000) Biochemistry & Molecular Biology of Plants

  6. Overall phylogeny of HRGPs Kieliszewski and Lamport 1994

  7. Motifs PYYPPH and TPVYK P hydroxylated to Hyp (O) 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)

  8. 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)

  9. 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-Hyp-Hyp-Hyp-Hyp-Tyr-Tyr-Tyr-Lys • More Tyr cross-linking possibilities Smith et al. 1986

  10. Purifying Extensin • Monomers soluble until incorporated into wall, how to purify? • SolubilizeHyp (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

  11. Purifying extensin, pectin cross-link Digests homogalacturonan Remove cellulose and XyG Remove furanosyl linkages (Ara) Remove ionically associated polymers RG-I still present Remove all sugars RG-I remains in an insoluble fraction, suggesting covalent linkage to pectin

  12. 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) • Extensinpectate • New model for wall assembly

  13. ExtensinPeroxidase (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)

  14. 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

  15. 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)

  16. di-IDT and pulcherosine: Intermolecular cross-linking Brady and Fry, 1998

  17. 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

  18. 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 Department of Biochemistry and Molecular Biology, University of Massachusetts Department of Chemistry and Biochemistry, Ohio University Department of Biology and Environmental Science, University of Sussex, UK

  19. The RSH mutant • -root, -shoot, -hypocotyl defective • Identified by Hall and Cannon 2002 • Major developmental problems • Severely misshapen cells • Misplacement of cell plate

  20. Extensins in Arabidopsis • 20 homologous genes, but this one knockout has lethal phenotype!

  21. rsh/rshphenotype Heart stage embryos Root sections C-F rsh/rshphenotype includes incomplete (floating, hanging) walls and wall stubs

  22. 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 extensinarabinooligosaccharides • Deglycosylation by HF and sequencing confirmed identity as AtEXT3

  23. Protein Sequence Highly periodic. 11 identical 28-residue repeats, monomer = 308 amino acids

  24. Cross-linking Assay • Pulcherosine is more prevalent than di-Idt as the cross-linking tyrosine derivative

  25. 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?

  26. 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)

  27. C-terminal significance • GFP fused to C-terminus did not rescue rsh/rshdouble mutant • N-terminal fused GFP yielded functional RSH

  28. 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

  29. 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

  30. 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 self-recognition 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 extensinpectate 9.Extensin scaffolds template for orderly pectic matrix

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