There are many ankyrin-repeat proteins in plant cells. However, the distribution and function of these proteins are mostly unclear. By reverse transcription-polymerase chain reaction, a gene encoding an ankyrin-like protein was cloned from Arabidopsis and named AtANK1 （GenBank accession no. NM_120340）. The 6-His-tagged AtAnkl-N fusion protein was affinity-purified and its rabbit polyclonal antibody was obtained. Immuno-blotting with the purified anti-AtAnkl-N polyclonal antibody revealed that the relative molecular weight of the AtANK1 protein was about 76 kDa. By immunofluorescence labeling and immuno-gold labeling with the purified anti-AtAnkl-N polyclonal antibody, coupled with confocal and transmission electron microscopy observation. AtANK1 was found to be distributed on the membrane of the endoplasmic reticulum in Arabidopsis cells. Based on these results, we suggested that AtANK1 might be involved in endoplasmic reticulum-related protein localization and sorting in plant cells.
GRIP domain proteins, locating to the trans-Golgi network, are thought to play an essential role in Golgi apparatus trafficking in yeast and animal cells. In the present study, AtGRIP cDNA was amplified by reverse transcriptase PCR from RNA isolated from Arabidopsis seedling. The GST fusion protein of AtGRIP was affinity-purified and its rabbit polyclonal antibody was obtained. Immuno-blotting with the purified anti-AtGRIP polyclonal antibody demonstrated that the molecular mass of AtGRIP protein is about 92 kD, and its expression is not tissue-specific in Arabidopsis. Immunoflourescent labeling and confocal microscopy revealed that the AtGRIP protein was co-localized with Golgi stacks in Arabidopsis root cells. Immuno-gold labeling and electron microscopy observation showed that AtGRIP protein was mainly located to the membrane of the secretory vesicles of trans-Golgi network in Arabidopsis root cap cells. Taken together, these results indicate that the localization of GRIP domain proteins between plants and animal cells are conserved. These results also suggest that the AtGRIP may be involved in regulating the formation or sorting of Golgi-associated vesicles in plant cells.
