Distribution And Function Of Kinesin-13A And Ankyrin-like Protein AtANK1 In Plant Cells

Golgi apparatus is a central organdie for trafficking pathway in cell. In plant cells, it is generally assumed that actin-microfilament system plays the main role in the localization, organization and function of the Golgi apparatus. However, it is undetermined whether microtubule and its motors take part in the organization and function of the Golgi apparatus in plant cells.Kinesin-13A is an internal motor kinesin. It is co-localized with Golgi stacks in different cells of Arabidopsis, implying that it may participate in controlling the structure and function of Golgi apparatus in plant cells (Lu et al, 2005). However, its precise localization and function on the Golgi apparatus is unclear. By immuno-gold labeling, coupled to transmission electron microscopy, we found that AtKinesin-13A existed mainly on the membrane of Golgi vesicles in Arabidopsis cells. By T-DNA insertional AtKinesin-13A mutant, the number and diameter of Golgi vesicles is sharply decreased in root-cap secretory cells of Arabidopsis, the root-cap secretory cells are vacuolization, and the number of root border cells is reduced markedly. Additionally, by draughting treatment for 10 days, the AtKinesin-13A mutant is more sensitive to the drought stress than the wild-type, and is dead earlier. Our results suggest that AtKinesin-13A may involve in regulating the formation and maintenance of plant Golgi apparatus secretory vesicles. The vacuolization of root-cap secretory cells and much more sensitivity of the mutant line to the drought stress may be the indirect results of AtKinesin-13A mutation。Using degenerated RT-PCR and immunoblotting with anti-AtKinesin-13A antibody, AtKinesin-13A-like protein, NtKinesin-13A, also was found to exist in tobacco, and its molecular weight is about 120 kDa. It can be expressed in most organs of tobacco. Bioinformatics analysis indicated that NtKinesin-13A-like genes also exist in rice. These results indicate that kinesin-13A is ubiquitous in plant cells. By immunoflorecence labeling and immuno-gold labeling, coupled to cofocal and transmission electron microscopy, we found NtKinesin-13A also bound to the membrane of Golgi vesicles. The similar localization between AtKinesin-13A and NtKinesin-13A implies that they have similar biological function.Ankyrins were found firstly as the main element of membrane skeleton in animal cells. Their main structural property is the tandem repeated ANK repeat. The number of the ANK repeat is different in diverse ankyrin isoforms. Subsequently, the membrane skeleton constituted of spectrin and ankyrin exists under most of membranes, especially the Golgi membrane skeleton which is paid more attention. However, it is uncertain whether the Golgi membrane skeleton is present in plant cells. Up to now, ankyrin-like protein related with membrane skeleton has not been discovered in plant cell. In addition, there are 105 genes encoding ANK repeat containing protein in Arabidopsis genome, and most of them are not studied.By RT-PCR, we cloned a gene encoding an ankyrin-like protein from Arabidopsis total RNA, and this gene is named AtANK1. The result of immunoblotting revealed the molecular weight ofAtANKl is about 70kDa. The AtANKl is the most similar ANK repeat containing protein to 119kDa ankyrin (AnkG₁₁₉) of human. AnkG₁₁₉ co-localizes with ER, βⅢspectrin and COP I , suggesting that it may involved in the organization of endomembrane system. By immunoflorecence labeling and immuno-gold labeling, coupled to cofocal and transmission electron microscopy, we found AtANK1 distributed to the outer-membrane of ER in Arabidopsis cells. Based on these results, we suggest that AtANKl may involve in the organization of ER domain or substance exchange between ER and Golgi in Arabidopsis cells.