Analysis of the Vacuolar Luminal Proteome of Saccharomyces Cerevisiae Jean-Emmanuel Sarry1*, Sixue Chen2*, Richard P
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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 Despite its large size and the numerous processes in which it is implicated, 2D gel electrophoresis; luminal proteins; neither the identity nor the functions of the proteins targeted to the yeast mass spectrometry; proteome; vacuole vacuole have been defined comprehensively. In order to establish a method- purification ological platform and protein inventory to address this shortfall, we refined Correspondence techniques for the purification of ‘proteomics-grade’ intact vacuoles. As P. A. Rea, Plant Science Institute, confirmed by retention of the preloaded fluorescent conjugate glutathione– Department of Biology, Carolyn Hoff Lynch bimane throughout the fractionation procedure, the resistance of soluble Biology Laboratory, 433 South University proteins that copurify with this fraction to digestion by exogenous extra- Avenue, University of Pennsylvania, vacuolar proteinase K, and the results of flow cytometric, western and mar- Philadelphia, PA 19104, USA ker enzyme activity analyses, vacuoles prepared in this way retain most of Fax: +1 215 898 8780 their protein content and are of high purity and integrity. Using this mate- Tel. +1 215 898 0807 E-mail: [email protected] rial, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of *These authors contributed equally to this these, 260 were identified by peptide mass fingerprinting and peptide work sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified (Received 28 March 2007, revised 30 May in this way, many of which correspond to alternate size and charge states 2007, accepted 27 June 2007) of the same parent translation product, can be assigned to 117 unique doi:10.1111/j.1742-4658.2007.05959.x ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacu- olar proteins of unknown function, or other proteases, glycosidases, lipid- binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar prepara- tions, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and ⁄ or salvage purposes. The vacuole of the budding yeast Saccharomyces cere- processes ranging from macromolecule degradation visiae, which can occupy as much as 25% of the total and salvage, pH and general ion homeostasis, osmo- intracellular volume, participates in numerous cellular regulation and volume regulation, to the storage of Abbreviations APE1, aminopeptidase I; APE3, aminopeptidase Y; BLH1, bleomycin hydrolase; 2-DE, 2D gel electrophoresis; ER, endoplasmic reticulum; GSH, glutathione; HSP, heat shock protein; LC, liquid chromatography; PRB1, vacuolar protease b; PRC1, carboxypeptidase Y; QTOF, quadrupole time-of-flight; SGD, Saccharomyces Genome Database; SOB, sorbitol buffer; SUC, sucrose buffer. FEBS Journal 274 (2007) 4287–4305 ª 2007 The Authors Journal compilation ª 2007 FEBS 4287 Proteomics of yeast vacuolar lumen J.-E. Sarry et al. amino acids, carboxylic acids, carbohydrates and some of the vacuoles of other fungal systems and plants, vitamins, and the sequestration of endogenous and and also the lysosomal compartments of animal cells. exogenous toxins. Given this degree of multifunctional- Here we describe refinement of a procedure to purify ity, what is perhaps surprising is how little is known of intact vacuoles from yeast and elucidate the identity of the range of proteins found in this compartment and the protein species found in the lumen by the com- the types of modifications to which they are subject. bined application of 2D gel electrophoresis (2-DE), The vacuole is known to be a major site for protein MALDI-TOF-MS and liquid chromatography (LC) turnover in the yeast cell. As such, several alternate coupled to electrospray ion trap or quadrupole time- but overlapping protein-transport pathways converge of-flight (QTOF) tandem MS. In so doing, we establish on this organelle [1–3]. Several canonical vacuolar pro- a methodological platform for defining the proteome teases, as newly synthesized proteins, are transported of the vacuolar lumen of S. cerevisiae and a compre- via the secretory pathway to the endoplasmic reticulum hensive data set containing polypeptide species that (ER) lumen or membrane, transit from the ER to the may be subjected to further functional characteriza- Golgi apparatus, and in the late Golgi are diverted to tion. the vacuole. Other proteins, as exemplified by alkaline To date, there have been three published analyses of phosphatase, enter the vacuole via the so-called ‘alka- the vacuolar proteome in the model plant Arabidopsis line phosphatase’ pathway by the direct fusion of thaliana. Two of these focus exclusively on the vacuo- Golgi-derived vesicles, or in the case of those coming lar membrane [5,6], and the third, which examines from the cell surface, are delivered to the vacuole luminal as well membrane proteins [7], though seminal, endocytotically by the formation of multilamellar is difficult to assess and put into context for two rea- bodies that are released into the vacuole lumen. Oth- sons. First, it does not systematically address the ers, specifically some cytoplasmic proteins, enter the purity of the luminal protein fraction or take special vacuole by cytoplasm-to-vacuole targeting which over- precautions to guard against contamination by non- laps with the autophagic pathway, the mechanism vacuolar luminal proteins, so compromising assessment responsible for the nonselective delivery of cytosolic of the tightness of association of the proteins in ques- proteins and organelles in their entirety to the vacuole tion with the vacuolar compartment. Second, it is for degradation under stress conditions. based exclusively on the results of nonquantitative Despite this wealth of information on the mecha- shotgun approaches: multidimensional LC-tandem MS nisms of protein trafficking into the vacuole there is a or the combination of 1D gel electrophoresis-coupled lack of fundamental systems-level knowledge of the LC-MS. As a result, there is little or no information range of proteins found in this compartment. This on the relative levels of the proteins identified or a may pose an impasse for the rational analysis of many 2-DE-based map to which other researchers, who do cellular processes and ultimately cellular engineering. If not have immediate access to MS resources, might vacuolate cells, for instance those of yeast, are to be refer. Here, we present investigations directed at over- manipulated for enhanced nutritional quality, the pro- coming limitations of this type and assembly of the vision of pharmaceuticals or their precursors, the first protein map of the yeast vacuolar lumen. provision of precursors for manufacturing purposes, or environmental remediation applications, ready access Results and Discussion to a luminal proteomics toolbox detailing the protein profile of the vacuole lumen and how the latter is Assessment of vacuolar purity and integrity established and maintained by intravacuolar reactions and vacuolar protein-trafficking pathways is critical. Intact vacuoles were routinely purified from S. cerevi- S. cerevisiae is a model system for the identification siae strain SEY6210 using a procedure based on those and definition of eukaryotic protein functions because described by Wiemken et al. [8], Roberts et al. [9] and it is especially easy to manipulate molecularly and pos- Kim et al. [10], in which lysed spheroplasts were sub- sesses only a moderate number of ORFs, most of jected to multiple cycles of density-gradient and flota- which are devoid of the interpretatively complicating tion centrifugation on sorbitol–sucrose and Ficoll step introns found in the genomes of most other eukaryotes gradients. As determined by flow cytometry, fluores- [4]. Moreover, because it is vacuolate and the core cence microscopy, western blot analysis and marker machinery for protein delivery into and processing enzyme analyses, this procedure yields intact vacuoles within the vacuole is likely conserved in other vacuo- of high purity. lysosomal structures [3], investigations of S. cerevisiae The integrity of the final vacuole preparation was have the potential to contribute to our understanding assessed by comparison of its forward-scatter plots 4288 FEBS Journal 274 (2007) 4287–4305 ª 2007 The Authors Journal compilation ª 2007 FEBS J.-E. Sarry et al. Proteomics of yeast vacuolar lumen A total signal. Whereas the unfractionated spheroplast preparation consists of two components, the smaller of which likely corresponds to vacuoles that have under- gone premature liberation