Minisymposium 26: Proteomics

Abs # 47005: Vacuomics: quantitative definition of the vacuolar proteome of yeast and Arabidopsis

Presenter:	Collum, Richard P., rcollum@sas.upenn.edu
Authors	Collum, Richard P. (A)   Chen, Sixue  (A)   Sarry, Jean-Emmanuel  (A)   Liang, Shun  (A)   Peng, Mingsheng  (A)   Su, Yan-Hua  (A)   Yuan, Chao-Xing  (B)   Klionsky, Daniel J. (C)   Rea, Philip A. (A)
Affiliations:	(A): Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA (B): Proteomics Core Facility, University of Pennsylvania, PA 19104, USA (C): Department of Biology, University of Michigan, Ann Arbor, MI 48109, USA
Web Site:	http://www.bio.upenn.edu/faculty/rea

In a typical mature plant cell, the vacuole constitutes some 40-70% of total intracellular volume, serving as an intracellular warehouse or landfill for the storage and sequestration of a broad range of compounds. Despite its importance, neither the full complement of proteins targeted to this organelle nor its full range of functions have been defined. Given its volume and capacity for the accumulation of organic compounds of intermediate complexity, an understanding of vacuolar function will be crucial if plants are to be engineered for enhanced nutritional value and/or for the manufacture of pharmaceuticals or their precursors. To address this deficiency in our knowledge and understanding, we have refined techniques for the purification of “proteomics-grade” intact vacuoles inclusive of their luminal protein complement from the yeast Saccharomyces cerevisiae and plant Arabidopsis thaliana. To date, we have been able to clearly separate 270-300 polypeptide species from the vacuolar lumen of yeast by 2-DE and identify 52 of them unambiguously by MALDI-TOF-MS and LCQ-MS. Equivalently, we have resolved 250 and unambiguously identified 42 polypeptide species from the luminal proteome of Arabidopsis. Current efforts are directed at the development of generally applicable techniques based on proteinase K protection experiments and isotope-coded affinity tagging (ICAT) for elucidating the intracellular localization of soluble proteins on the basis of proteinase resistance and isotopic enrichment. Coupling of these techniques with the results of more traditional marker enzyme assays is crucial for assessing the organelle association of the proteins identified hand in hand with the enumeration of organellar enrichment. Since the core machinery for vesicle-mediated protein delivery to the vacuole is likely conserved in so far as several genome projects have revealed homologs of the components of the yeast trafficking pathways, these investigations will inevitably facilitate rational analyses of many vacuolysosomal systems. This work is funded by a Penn Genomics Institute Seed Grant and by NSF Grant No. MCB-0313461 awarded to P.A.R.