The Cellulose Synthase Superfamily

Abstract

The availability of a nearly complete genome sequence for Arabidopsis has created many novel opportunities to identify, by computational methods, the genes that encode enzymes, which have been difficult to characterize by conventional means. We have used this approach to identify a large family of genes of unknown function that show sequence similarity to cellulose synthase. Our working hypothesis is that these genes encode enzymes that catalyze the synthesis of non-cellulosic polysaccharides (Cutler and Somerville, 1997). Unrooted, bootstrapped tree of theCesA superfamily. ClustalX (version 1.8) was used to create an alignment of the full-length, publicly available protein sequences that was then bootstrapped (n = 5,000 trials) to create the final tree. Subfamilies are boxed. At, Arabidopsis; Gh, cotton; Le, tomato; Mt, Medicago truncatula; Os, rice; Pt, Populus tremuloides; Pt/Pa, Populus tremula × Populus alba. It is not known at this time whether other polypeptides are also required for cellulose synthase activity (i.e. the CesA polypeptides may be a component of a multisubunit enzyme complex). Until this matter is resolved we consider it expedient to simply refer to the CesA family members as cellulose synthase. The observation that IXR3(AtCesA7), which is required for secondary wall cellulose synthesis, is in a different branch of the CesA tree than RSW1(AtCesA1), which is required for primary wall synthesis (Fig. 1), may indicate that there is sequence divergence between the enzymes involved in primary and secondary wall synthesis. Reiterative database searches using the Arabidopsis Rsw1 (AtCesA1) and the cotton CesA polypeptide sequences as the initial query sequences revealed a large superfamily of at least 41CesA-like genes in Arabidopsis. Based on predicted protein sequences, we have grouped these genes into seven clearly distinguishable families (Fig. 1): the CesA family, which includes RSW1 and IRX3 (AtCesA7), and six families of structurally related genes of unknown function designated as the “cellulose synthase-like” genes (CslA,CslB, CslC, CslD, CslE, andCslG). The nomenclature for these families is still under discussion (http://mbclserver.rutgers.edu/CPGN/CelluloseWeb/CesA.proposal.html), so the Csl designation for these genes should be considered temporary and may be revised as the enzymatic function of the members of each family is determined. All of the members of the cellulose synthase superfamily appear to be integral membrane proteins, with three to six transmembrane domains in the carboxy terminal region of the protein and one or two transmembrane domains in the amino terminal region. It is thought that the CesA proteins are located in the plasma membrane (Delmer, 1999). If the Csl proteins participate in the synthesis of non-cellulosic polysaccharides, they would be expected to be located in the Golgi apparatus. Preliminary analysis of CslB, CslG, and CslE fusions to green fluorescent protein appear to localize to the Golgi (T. Richmond and C. Somerville, unpublished data). Also, immunolocalization studies with an antibody to the CslA protein indicates that this family is localized to the cytoplasm (i.e. the Golgi apparatus) rather than the plasma membrane (N. Sprenger and C. Somerville, unpublished data). Comparison of the gene structure of representative genes of the Arabidopsis CesA superfamily. Colored boxes represent exons and the lines connecting them denote introns. Thick vertical black bars indicate predicted transmembrane domains as predicted by HMMTOP (http://www.enzim.hu/hmmtop/). Thin blue bars represent conserved Asp residues, and the thicker gray bar represents the QxxRW domain. Thin lines connecting different genes indicate conserved intron-exon junctions. All members of the CesA family contain a putative LIM-like Zn-binding domain/RING finger domain in the N-terminal region, which is similar to several putative plant Leu zipper transcription factors (Kawagoe and Delmer, 1997a, 1997b; Arioli et al., 1998). LIM domains are known to mediate protein-to-protein interactions (Bach, 2000), whereas RING finger domains are thought to play a role in ubiquitin-mediated proteolysis (Freemont, 2000). These domains may play a role in mediating CesA function via protein partners or targeted degradation. All of the Csl proteins lack this amino terminus extension, including the CslD family, which contains proteins similar in size to the CesAs. Although the various CesA and Csl proteins vary in their degree of sequence similarity to one another (TableI), they share several features that have been proposed to be indicative of processive glycosyltransferases (Saxena et al., 1995). All of the CesA and Cslgene products contain a D,D,D,QxxRW motif (Fig. 2), which has been proposed to define the nucleotide sugar-binding domain and the catalytic site of these enzymes. Based on this motif, the proposed topology of these proteins (discussed above), and sequence-based classification, the vario