Chimeric Proteins Suggest That the Catalytic and/or C-Terminal Domains Give CesA1 and CesA3 Access to Their Specific Sites in the Cellulose Synthase of Primary Walls

Abstract

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3WT protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3 rsw5 ) does not.

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1 A903V and CESA3 T942I of cellulose synthase

“…It is possible that for both CBIs the site of inhibition occurs in this region. The C-terminal transmembrane spanning region has been proposed to mediate subunit association in the CSC (38), and therefore a CBI targeting this region could inhibit CSC formation. Supporting this postulate, live-cell imaging studies revealed clearance of YFP∷ CESA6 from the PM after quinoxyphen treatment, although resistance was conferred by CESA1 A903V .…”

Complexes with Mixed Primary and Secondary Cellulose Synthases Are Functional in Arabidopsis Plants

“…The C-terminal region contains two strongly conserved Cys residues, and we speculate that the formation of disulfide bonds between the C terminus of one CESA and one of the other Cys-rich regions in another CESA might help mediate complex assembly. Chimeric CESA and CESA/CSLD proteins exchanging the N-terminal region (Wang et al, 2006) and catalytic domain (Park et al, 2011) have both retained the identity of the genetic position or localization of the C-terminal domain. This site was absolutely conserved in CESA families 3, 4, 6, and 7 but not in CESA families 1 and 8, with CESA families 3 and 7 showing more similarity to each other than with the other CESAs (Carroll and Specht, 2011).…”

“…In order to further analyze the similarities and differences between the primary and secondary CESAs making up the complex, the sites of the C-terminal rsw5 mutation implicated in disrupting the incorporation of CESA3 into the primary cellulose synthase complex were compared (Wang et al, 2006;Carroll and Specht, 2011). The C terminus is a putatively cytosolic region of approximately 20 amino acids that follows the eighth transmembrane domain.…”

Cellulose Synthesis and Its Regulation

“…Since CESA1 and CESA3 are essential components in the CSC and CESA1 and CESA3 share the highest amino acid similarity, it is reasonable to speculate that CESA1 and/or CESA3 may reside in a special position in the CSC. A chimeric study of CESA1 and CESA3 indicated that these two CESAs have specific positions within the CSC (Wang et al, 2006).…”

“…Col (Brown, 1999b;CanoDelgado et al, 2003) eli1-2 A522V Col (Brown, 1999b;CanoDelgado et al, 2003) rsw5 P1056S temperature sensitive, radial swelling, dwarf plants, cellulose deficiency Col (Wang et al, 2006) thanatos P578S semi-dominant allele, short, swollen root and hypocotyl, cellulose deficiency Col (Daras et al, 2009) mre G916E short, swollen root and etiolated hypocotyl, cellulose deficiency, dwarf plants Col (Pysh et al, 2012) CESA4 (At5g44030) irx5-1 thin, irregular xylem and interfascicular cell walls, cellulose content deficiency, dwarf plants Ler irx5-2 W995stop Ler irx5-3 Q263stop Ler exi2 Y939stop small rosette leaves, flowers, siliques, reduced cell elongation, dwarf plants, altered vasculature and cell morphology, cellulose content deficiency, impaired water transport…”