Poster: Proteomics

Abs # 961: A proteomic approach for the identification of transporters and channels of Arabidopsis thaliana

Presenter:	Keta, Sumie , i022003d@mbox.media.nagoya-u.ac.jp
Authors	Keta, Sumie  (A)   Shiratake, Katsuhiro  (A)   Sazuka, Takashi  (B)   Shibata, Daisuke  (B)   Maeshima, Masayoshi  (A)   Yamaki, Shohei  (A)
Affiliations:	(A): Graduate school of Bio-agricultural Sciences, Nagoya University (B): Kazusa DNA Research Institute

A proteomic approach was developed in order to identify transporters and channels especially localized in vacuolar membrane of Arabidopsis thaliana. About 4-week-old plants were homogenized and vacuolar membrane was enriched by combination of several sucrose density gradient centrifugations. The vacuolar membrane in the final fraction was enriched about 5-fold compared with microsome. The membranes were washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimentional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reversed-phase high-performance liquid chromatography. Among 163 peptide peaks analyzed by Edman sequencing, 64 peptide sequences were informative. A total of 34 proteins were identified definitively with a protein-specific sequence. They included 13 transporters and 3 channels. They also included some multi-membrane spanning proteins whose functions were unclear. These results show this approach was effective to identify transporters and channels. The membrane fraction used in this experiment contained not only vacuolar membrane but also other ones. Thus, certain intracellular localizations of the identified proteins were unclear. To determine them, we are preparing antibodies against those proteins, now.