tailieunhanh - Báo cáo khoa học: Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae

Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a method-ological platform and protein inventory to address this shortfall, we refined techniques for the purification of ‘proteomics-grade’ intact vacuoles. | ỊFEBS Journal Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae Jean-Emmanuel Sarry1 Sixue Chen2 Richard P. Collum1 Shun Liang1 Mingsheng Peng1 Albert Lang1 Bianca Naumann1 Florence Dzierszinski1 Chao-Xing Yuan3 Michael Hippler1 and Philip A. Rea1 1 Department of Biology University of Pennsylvania Philadelphia PA USA 2 Department of Botany Genetics Institute University of Florida Gainesville FL USA 3 Proteomics Core Facility University of Pennsylvania Philadelphia PA USA Keywords 2D gel electrophoresis luminal proteins mass spectrometry proteome vacuole purification Correspondence P. A. Rea Plant Science Institute Department of Biology Carolyn Hoff Lynch Biology Laboratory 433 South University Avenue University of Pennsylvania Philadelphia PA 19104 USA Fax 1 215 898 8780 Tel. 1 215 898 0807 E-mail parea@ These authors contributed equally to this work Received 28 March 2007 revised 30 May 2007 accepted 27 June 2007 doi Despite its large size and the numerous processes in which it is implicated neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall we refined techniques for the purification of proteomics-grade intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathionebimane throughout the fractionation procedure the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extra-vacuolar proteinase K and the results of flow cytometric western and marker enzyme activity analyses vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these 260 were identified by peptide