Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein-TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

The formation of the ascospore wall of Saccharomyces cerevisiae requires the coordinate activity of enzymes involved in the biosynthesis of its components such as chitosan, the deacetylated form of chitin. We have cloned the CDA1 and CDA2 genes which together account for the total chitin deacetylase activity of the organism. We have shown that expression of these genes is restricted to a distinct time period during sporulation. The two genes are functionally redundant, each contributing equally to the total chitin deacetylase activity. Diploids disrupted for both genes sporulate as efficiently as wild type cells, and the resulting mutant spores are viable under standard laboratory conditions. However, they fail to emit the natural fluorescence of yeast spores imparted by the dityrosine residues of the outermost ascospore wall layer. Moreover, mutant spores are relatively sensitive to hydrolytic enzymes, ether, and heat shock, a fact that underscores the importance of the CDA genes for the proper formation of the ascospore wall.The cell wall of Saccharomyces cerevisiae, in spite of its apparent rigidity, is a highly dynamic structure. Its general biological functions include mechanical protection, determination of cell shape, and modulation of selective uptake of molecules. Moreover, yeast are able to alter the composition and/or the structure of their cell wall throughout the different stages of their life cycle such as budding, mating, and sporulation. Such plasticity requires the coordinated regulation of the expression of genes involved in the formation of the cell wall. Although cell wall assembly and its regulation have been extensively studied during vegetative growth (1-3), there is limited information on the composition and formation of the more complex ascospore cell wall.Sporulation is the developmental stage that diploid cells enter when starved for nitrogen and a fermentable carbon source. It proceeds with meiosis followed by the encapsulation of the four haploid nuclei within the spore wall. This wall offers increased protection to stress conditions as compared to the wall of vegetative cells (4) and consists of four layers (5, 6). The two inner layers are formed by closely juxtaposed glucans and mannans and appear as a single layer very similar in morphology to the vegetative cell wall (6, 7). The outermost layer consists of an insoluble macromolecule, probably a protein, which contains a high number of cross-linked tyrosine residues (8 -10). Between these layers a chitosan layer has been identified (6). Chitosan, a ␤-(134)-D-glucosamine homopolymer, was initially detected in the cell wall of Zygomycete species (11) and is produced by the deacetylation of nascent chains of chitin, a ␤-(134)-N-acetyl-D-glucosamine homopolymer produced by the action of chitin synthases (12-14). The deacetylation reaction is catalyzed by the enzyme chitin deacetylase (CDA) 1 (15, 16).Chitin deacetylases are involved either in the formation of the cell wall (12,14,17) or in the deacetylation of chitin oligosaccharides ...