1

EMBnet Course Proteomics Using Bioinformatics Tools

2008, Basel Practical Course

2

1. Purpose of the Practical Part The purpose of the practical part of the EMBnet course is to give the participants a basic training in performing proteomic experiments. For this, we will do a set of classical experiments with the yeast protein kinase Npr1 (see Introduction below). You will learn how to do a simple protein digestion, how to interpret peptide mass spectra obtained from protein digests and how to look for protein phosphorylation. You will see how phosphopeptides can be selectively enriched and how relative changes that occur in phosphorylation can be quantitated. Also, to obtain a feel for how the stability of the Npr1 kinase is regulated you will try to map potential breakdown products of the kinase by classical in-gel digestion procedures. Those only interested in the nitty-gritty of the experiments, the introduction can be skipped down to ‘The Npr1 Protein Kinase’ part. For those interested in the biology that is behind the experiments, here’s a brief introduction into nitrogen metabolism of yeast and into the function of the Npr1 kinase. 2. Introduction into Nitrogen Metabolism in Yeast Growth and proliferation in micro organisms depend on two nutritional inputs: a carbon and a nitrogen source. Yeast cells can assimilate a variety of carbon and nitrogen compounds but if the cells grow in media containing alternative sources they discriminate between preferred and non-preferred sources. Therefore, to optimally use the available nutrients, yeast cells respond to the quality of the carbon and nitrogen source present in the environment. Depending on the availability of nutrients, the cells adapt their metabolic and transcriptional program to provide the proper set of proteins responsible for uptake, transport, and metabolism of nutrients. In addition, cells execute the appropriate developmental program like vegetative or filamentous growth, or cell cycle arrest and mating. Thus, the choice of the developmental program depends on the quantity and quality of the nutrients. 2.1. Nitrogen Regulation Yeast cells require nitrogen to produce amino acids for protein synthesis and building blocks for RNA and DNA synthesis. The amino nitrogen of glutamate serves as the source of 85% of the total cellular nitrogen whereas the amide group of glutamine is the source of the remaining 15% [1]. For the assimilation of nitrogen, permeases are required and metabolic enzymes for the production of ammonia from nitrogen containing compounds. Ammonia reacts with α-ketoglutarate that is provided by the citric acid cycle to produce glutamate. This reaction is catalyzed by glutamate dehydogenase (GDH1) [2]. In an ATP dependent process, glutamine synthetase (GLN1) catalyzes the production of glutamine from glutamate and ammonia [3]. Yeast cells can grow in different nitrogen media. They discriminate between good (preferred) and poor (non-preferred) nitrogen media. S. cerevisiae, like

3

most micro organisms, transports, accumulates, and processes good nitrogen sources in preference to poor ones. The general response to the quality of nitrogen source is achieved by nitrogen catabolite repression (NCR) where enzymes and permeases for the utilization of non-preferred compounds are repressed when preferred nitrogen sources are available [4]. This is achieved by repressing the transcription of genes involved in the utilization of non-preferred nitrogen sources, but also by post-translational regulation like intracellular sorting of specific permeases. Nitrogen sources that do not derepress pathways for the utilization of alternative nitrogen sources are preferred nitrogen sources.

Fig. 1: Nitrogen metabolism. Glutamate and glutamine serve as building blocks for the cellular synthesis of amino acids, DNA and RNA. Glutamate is synthesized from α-ketoglutarate and ammonia. In turn, glutamine is produced from glutamate and ammonia. Nitrogen containing compounds like urea, proline, and arginine are fed into the pathway at intermediate points [5]. 2.2. Yeast Nitrogen Permeases Yeast encodes over 250 membrane transporters which are responsible for the selective transport of nutrients [6]. Among them are 19 amino acid transporters with distinct substrate specificity, affinity, and transport capacity. The amino acid permeases are integral membrane proteins with 12 predicted transmembrane domains and are delivered to the plasma membrane via the secretory pathway. They are responsible for the uptake of amino acids, polyamines, and choline from the environment for protein synthesis and for use as sources of nitrogen [7, 8]. The permeases can be divided into two classes according to their regulation and function. One group of permeases is repressed during growth on a preferred nitrogen source and coordinately derepressed if the cells are shifted to a poor nitrogen source. Representatives of this group are the general amino acid permease Gap1, which transports all naturally occurring amino acids [9], and Put4, which transports only proline [10, 11]. These permeases are important to provide the cells with nitrogen containing compounds that are fed into the production of glutamate and glutamine [12, 13]. The other class includes permeases that are present at the

4

plasma membrane when cells grow on a good nitrogen source. Some of them are down regulated when the cells are shifted to a poor nitrogen source. Members of this class are mostly specific for particular amino acids, or chemically related compounds, such as the histidine permease Hip1 [14], the basic amino acid permease Can1 [15], and Tat2, a tryptophan permease [16]. The amino acids taken up by these permeases are destined directly for protein synthesis [5]. 2.3. Nitrogen Sensing A prerequisite for a proper physiological response is the ability to sense and transduce information regarding the quality of nitrogen sources present in the extra- and intracellular environment. Nitrogen sensing and signal transduction pathways in yeast consist of the extracellular amino acid sensor SPS, the extracellular ammonia sensor Mep2 and the intracellular nitrogen sensing systems involving Gcn2 and the TOR pathway (Fig. 2). The different pathways have distinct as well as overlapping functions.

Fig. 2: Overview of yeast signaling pathways that sense and transduce the availability of nitrogen in the environment [17]. The Tor (target of rapamycin) kinases were found to be central controllers of the nitrogen catabolite repression. TOR was originally identified genetically by mutations in yeast that acquired resistance to the growth-inhibitory properties of the immunophilin-immunosuppressant complex FKBP-rapamycin [18]. TOR is a phosphatidylinositolkinase-related protein kinase (PIKK) [19] that is conserved in all eukaryotes examined. Unlike all the other eukaryotes, yeast harbors two different TOR genes, termed TOR1 and TOR2 with 67% sequence identity [20]. Early genetic observations suggested that TOR1 and TOR2 have a shared function that is required for transit through G1 phase which is sensitive to rapamycin and TOR2 has an additional, unique function

5

that TOR1 is unable to perform. This essential function of TOR2 is insensitive to rapamycin [19-23].

Fig. 3: The two TOR complexes in Saccharomyces cerevisiae. The TOR complexes are shown as dimers with its associated proteins. TORC1 consists of TOR1 or TOR2, LST8, KOG1, and TCO89 and is positively regulated by nutrients and negatively by stress and rapamycin-treatment. TORC1 outputs that promote the accumulation of mass are depicted with black arrows, stress- and starvation-induced processes that TORC1 regulates negatively with red bars. TORC2 consists of TOR2, LST8, AVO1-3, and BIT61 and regulates actin organization. Upstream regulators of TORC2 are not known. The different domains of TOR (HEAT, FAT, FRB, Kinase, and FATC) are indicated [24].

The TOR-shared and TOR2-unique functions define two separate signaling pathways that are mediated by the two distinct multiprotein complexes TOR complex 1 (TORC1) and TOR complex 2 (TORC2) [25] (Fig. 4). TORC1 contains either Tor1 or Tor2 and the three associated proteins Lst8, Kog1, and Tco89 [25, 26]. TORC1 is inhibited by the FKBP-rapamycin complex. Disruption of TORC1 in yeast mimics the phenotype seen after rapamycin treatment, suggesting that TORC1 is the physiological target of rapamycin [25]. TORC1 positively controls protein synthesis at multiple levels like translation initiation, expression and assembly of the translation machinery, mRNA turnover, and the activity of high affinity amino acid permeases that pump amino acids for immediate use by the translation machinery [27]. In contrast TORC1 negatively regulates a number of stress-related functions like autophagy and the activities of different stress-responsive transcription factors [27]. TORC2 contains Tor2 together with the proteins Lst8, Avo1, Avo2, Avo3, Bit2, and Bit6 [25, 26, 28]. TORC2 fails to bind the FKBP-rapamycin complex which makes it insensitive to rapamycin [25]. TORC2 is important for the cell-cycle dependent polarization of the actin cytoskeleton to facilitate trafficking of macromolecules from the mother cell to the bud, the main place of growth [25]. Activation of mammalian TORC1 involves the tuberous sclerosis proteins TSC1 and TSC2 and the small GTPase Rheb which integrates signals from growth factors, nutrients, energy, and stress [24]. However, TORC1 in S.

6

cerevisiae responds to nutrients despite the absence of functional Rheb and TSC orthologs. Upstream regulators of TORC1 in yeast remain elusive. 2.3. Nitrogen-regulated Permease Sorting Upon nitrogen limitation or rapamycin treatment, high-affinity amino acid permeases such as Tat2 are rapidly internalised and sorted to the vacuole for degradation, while pre-existing Gap1 is delivered to the plasma membrane and stabilised [29]. The differential sorting of these two classes of permeases is mediated by the Ser/Thr kinase Npr1 [30-32]. Nitrogen limitation or rapamycin treatment activates Npr1 by dephosphorylation with the TOR-modulated Sit4 phosphatase [29, 33]. The TOR kinase promotes the association of the Sit4 phosphatase and the regulatory subunit Tap42 [34]. Upon nitrogen starvation or treatment with rapamycin, Sit4 is activated by dissociation from its inhibiting subunit Tap42. In turn, Sit4 dephosphorylates Npr1 and as a consequence, Gap1 is allowed to reach the plasma membrane (Fig. 1). Simultaneously, the high-affinity tryptophan permease Tat2 is rapidly endocytosed and targeted to the vacuole. On the other hand, shifting cells to a good nitrogen source causes Gap1 internalization and re-routing of pre-existing Gap1 from the Golgi to the vacuole without ever reaching the plasma membrane, whereas Tat2 is stabilised at the plasma membrane [35-37]. Gap1 inactivation is brought about by the E3 ubiquitin protein ligase Rsp5 together with the Rsp5 binding proteins Bul1 and Bul2 [36, 38, 39].

Fig. 4: In poor nitrogen medium (left panel), Tor is inactive leading to the release of the inhibitory subunit Tap42 from the phosphatase Sit4 which in turn dephosphorylates Npr1. Active Npr1 allows sorting of the general amino acid permease to the plasma membrane. In nitrogen-rich medium (right panel), Tor keeps Sit4 inactive by promoting the formation of the Sit4/Tap42 complex. PM, plasma membrane; Ub, ubiquitin; MVB, multivesicular body.

7

2.4. The Npr1 Protein Kinase

The Npr1 kinase is required for the optimal activity of several transport systems for nitrogenous compounds like the general amino acid permease Gap1, and the proline permease Put4. Sequence analysis of the NPR1 gene showed that it encodes an 86 kDa Ser/Thr protein kinase of 790 amino acid residues [31, 32]. The amino-terminal part of Npr1 (residues 17-413) comprises a serine-rich domain with 26% serine content whereas the carboxy-terminal part of Npr1 (residues 438-742) contains sequence motifs characteristic of the catalytic domain of protein kinases [32]. Interestingly, a WW domain-interacting PPXY motif can be found [40] at the C-terminal end of Npr1. Npr1 sequence analysis reveals three putative PEST motifs in Npr1. Two of them are located in the N-terminal domain (residues 33-51 and 73-105) and one is located around the PPXY motif (residues 680-691). PEST sequences are signals for protein instability. Phosphorylation within PEST sequences often specify their recognition and processing by the ubiquitination pathway [41].

Fig. 5: Domain structure of Npr1. The N-terminal domain is rich in serine, while the kinase domain lies in the C-terminal part of Npr1. The protein contains three PEST sequences that might be involved in regulating Npr1’s stability. The PEST sequence in the catalytic domain contains a PPXY motif that interacts with WW domains. For the EMBnet course, Npr1 is expressed as a GST fusion protein. Npr1 belongs to the fungus-specific Npr/Hal5 subfamily of protein kinases [42]. The members of this subfamily (Stk1/Stk2 and Hal4/Hal5) are highly conserved in the kinase domain, suggesting that these kinases are functionally related and that they are regulated in an analogous manner [43]. The kinases Stk1 and Stk2 are involved in polyamine transport [43], while Hal4 and Hal5 regulate the Trk1/2 potassium transporter activity in response to potassium starvation [44]. Recent results indicate that Hal4/Hal5 stabilize the Trk1 transporter at the plasma membrane under low potassium conditions by preventing their endocytosis and vacuolar degradation [45] similar to the Npr1 mediated stabilization of Gap1 at the plasma membrane. NPR1 transcription is independent of the quality of the nitrogen source [31]. Its activity is regulated through the TOR signalling pathway by means of phosphorylation [29]. Npr1 isolated from cells grown on ammonia is highly phosphorylated whereas rapamycin treatment leads to substantial dephosphorylation of Npr1 [33]. In nitrogen-poor media or upon rapamycin

8

treatment, Sit4 phosphatase is activated and dephosphorylates Npr1 [29]. As a consequence Gap1 is sorted to the plasma membrane where it is stabilized whereas Tat2 is rapidly endocytosed and targeted to the vacuole [29, 37, 46]. Rapamycin mimics nitrogen starvation by inhibiting the core of the multiprotein complex TORC1 [25]. Isolation of GST-Npr1 from yeast cells that had been grown in a nitrogen-rich medium results in a protein that migrates as a diffuse band on SDS gel electrophoresis. In addition, GST-Npr1 obtained from a good nitrogen source migrates slower than the well-focused band of phosphatase-treated GST-Npr1 (Fig. 6A). The mobility shift of GST-Npr1 following phosphatase treatment indicates substantial phosphorylation of GST-Npr1. Rapamycin treatment of cells grown in nitrogen-rich medium results in a GST-Npr1 band whose migration resembles the phosphatase-treated protein, although with considerable band broadning (Fig. 6A). Similar GST-Npr1 preparations are obtained from cells that had been grown in proline medium following treatment with glutamine (Fig. 6A). The shift was caused by treating the cells with 4 mM glutamine for 15 min before the cells were harvested and lysed. In vitro kinase assay with myelin basic protein (MBP) showed a substantial increase of substrate phosphorylation following rapamycin treatment. A decrease of substrate phosphorylation was observed by treating the proline-grown cells with glutamine. Autophosphorylation of GST-Npr1 remained unaffected by the treatment (Fig. 6B). Therefore, inactivation of Npr1 is brought about by phosphorylation, while dephosphorylation, either by pharmacological treatment with rapamycin or by growth in nitrogen poor medium, activates Npr1. Therefore, by mapping experiments, we would like to exploit the sites of phosphorylation in Npr1 and we would also like to exploit the degree to which Npr1 becomes dephosphorylated upon activation. Since Npr1 contains a protein-protein interaction motif, we would like to exploit its partners by proteomic screens.

9

Fig. 6: Npr1 is a glutamine-regulated protein kinase. A: Colloidal-blue stained GST-Npr1 isolated from yeast cells grown in nitrogen-rich (NH4

+) or nitrogen-poor medium (Pro). rapa, AP, gln indicates treatment with rapamycin, alkaline phosphatase, or glutamine, respectively. B: Colloidal-blue stained gel and autoradiograph of a kinase assay with GST-Npr1 and myelin basic protein (MBP) as substrate. MBP is marked with a triangle.

10

References 1 Cooper, T. G. (1982) Nitrogen metabolism in Saccharomyces cerevisiae. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY 2 Grenson, M., Dubois, E., Piotrowska, M., Drillien, R. and Aigle, M. (1974) Ammonia assimilation in Saccharomyces cerevisiae as mediated by the two glutamate dehydrogenases. Evidence for the gdhA locus being a structural gene for the NADP-dependent glutamate dehydrogenase. Mol Gen Genet. 128, 73-85 3 Mitchell, A. P. and Magasanik, B. (1983) Purification and properties of glutamine synthetase from Saccharomyces cerevisiae. J Biol Chem. 258, 119-124 4 Magasanik, B. (1992) Regulation of nitrogen utilization. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY 5 Magasanik, B. and Kaiser, C. A. (2002) Nitrogen regulation in Saccharomyces cerevisiae. Gene. 290, 1-18 6 Van Belle, D. and Andre, B. (2001) A genomic view of yeast membrane transporters. Curr Opin Cell Biol. 13, 389-398 7 Andre, B. (1995) An overview of membrane transport proteins in Saccharomyces cerevisiae. Yeast. 11, 1575-1611 8 Regenberg, B., During-Olsen, L., Kielland-Brandt, M. C. and Holmberg, S. (1999) Substrate specificity and gene expression of the amino-acid permeases in Saccharomyces cerevisiae. Curr Genet. 36, 317-328 9 Jauniaux, J. C. and Grenson, M. (1990) GAP1, the general amino acid permease gene of Saccharomyces cerevisiae. Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression. Eur J Biochem. 190, 39-44 10 Lasko, P. F. and Brandriss, M. C. (1981) Proline transport in Saccharomyces cerevisiae. J Bacteriol. 148, 241-247 11 Vandenbol, M., Jauniaux, J. C. and Grenson, M. (1989) Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases. Gene. 83, 153-159 12 Courchesne, W. E. and Magasanik, B. (1983) Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae. Mol Cell Biol. 3, 672-683 13 Wiame, J. M., Grenson, M. and Arst, H. N., Jr. (1985) Nitrogen catabolite repression in yeasts and filamentous fungi. Adv Microb Physiol. 26, 1-88 14 Tanaka, J. and Fink, G. R. (1985) The histidine permease gene (HIP1) of Saccharomyces cerevisiae. Gene. 38, 205-214 15 Hoffmann, W. (1985) Molecular characterization of the CAN1 locus in Saccharomyces cerevisiae. A transmembrane protein without N-terminal hydrophobic signal sequence. J Biol Chem. 260, 11831-11837 16 Schmidt, A., Hall, M. N. and Koller, A. (1994) Two FK506 resistance-conferring genes in Saccharomyces cerevisiae, TAT1 and TAT2, encode amino acid permeases mediating tyrosine and tryptophan uptake. Mol Cell Biol. 14, 6597-6606 17 Schneper, L., Duvel, K. and Broach, J. R. (2004) Sense and sensibility: nutritional response and signal integration in yeast. Curr Opin Microbiol. 7, 624-630 18 Heitman, J., Movva, N. R. and Hall, M. N. (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science. 253, 905-909 19 Cafferkey, R., Young, P. R., McLaughlin, M. M., Bergsma, D. J., Koltin, Y., Sathe, G. M., Faucette, L., Eng, W. K., Johnson, R. K. and Livi, G. P. (1993) Dominant missense mutations in a novel yeast protein related to mammalian

11

phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol Cell Biol. 13, 6012-6023 20 Helliwell, S. B., Wagner, P., Kunz, J., Deuter-Reinhard, M., Henriquez, R. and Hall, M. N. (1994) TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast. Mol Biol Cell. 5, 105-118 21 Helliwell, S. B., Howald, I., Barbet, N. and Hall, M. N. (1998) TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae. Genetics. 148, 99-112 22 Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R. and Hall, M. N. (1993) Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 73, 585-596 23 Zheng, X. F., Florentino, D., Chen, J., Crabtree, G. R. and Schreiber, S. L. (1995) TOR kinase domains are required for two distinct functions, only one of which is inhibited by rapamycin. Cell. 82, 121-130 24 Wullschleger, S., Loewith, R. and Hall, M. N. (2006) TOR signaling in growth and metabolism. Cell. 124, 471-484 25 Loewith, R., Jacinto, E., Wullschleger, S., Lorberg, A., Crespo, J. L., Bonenfant, D., Oppliger, W., Jenoe, P. and Hall, M. N. (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell. 10, 457-468 26 Reinke, A., Anderson, S., McCaffery, J. M., Yates, J., 3rd, Aronova, S., Chu, S., Fairclough, S., Iverson, C., Wedaman, K. P. and Powers, T. (2004) TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem. 279, 14752-14762 27 De Virgilio, C. and Loewith, R. (2006) Cell growth control: little eukaryotes make big contributions. Oncogene. 25, 6392-6415 28 Fadri, M., Daquinag, A., Wang, S., Xue, T. and Kunz, J. (2005) The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol Biol Cell. 16, 1883-1900 29 Schmidt, A., Beck, T., Koller, A., Kunz, J. and Hall, M. N. (1998) The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. Embo J. 17, 6924-6931 30 Grenson, M. (1983) Study of the positive control of the general amino-acid permease and other ammonia-sensitive uptake systems by the product of the NPR1 gene in the yeast Saccharomyces cerevisiae. Eur J Biochem. 133, 141-144 31 Vandenbol, M., Jauniaux, J. C., Vissers, S. and Grenson, M. (1987) Isolation of the NPR1 gene responsible for the reactivation of ammonia-sensitive amino-acid permeases in Saccharomyces cerevisiae. RNA analysis and gene dosage effects. Eur J Biochem. 164, 607-612 32 Vandenbol, M., Jauniaux, J. C. and Grenson, M. (1990) The Saccharomyces cerevisiae NPR1 gene required for the activity of ammonia-sensitive amino acid permeases encodes a protein kinase homologue. Mol Gen Genet. 222, 393-399 33 Jacinto, E., Guo, B., Arndt, K. T., Schmelzle, T. and Hall, M. N. (2001) TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. Mol Cell. 8, 1017-1026

12

34 Di Como, C. J. and Arndt, K. T. (1996) Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev. 10, 1904-1916 35 Roberg, K. J., Rowley, N. and Kaiser, C. A. (1997) Physiological regulation of membrane protein sorting late in the secretory pathway of Saccharomyces cerevisiae. J Cell Biol. 137, 1469-1482 36 Springael, J. Y. and Andre, B. (1998) Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae. Mol Biol Cell. 9, 1253-1263 37 Beck, T., Schmidt, A. and Hall, M. N. (1999) Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast. J Cell Biol. 146, 1227-1238 38 Helliwell, S. B., Losko, S. and Kaiser, C. A. (2001) Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease. J Cell Biol. 153, 649-662 39 Soetens, O., De Craene, J. O. and Andre, B. (2001) Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1. J Biol Chem. 276, 43949-43957 40 Ingham, R. J., Gish, G. and Pawson, T. (2004) The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene. 23, 1972-1984 41 Lanker, S., Valdivieso, M. H. and Wittenberg, C. (1996) Rapid degradation of the G1 cyclin Cln2 induced by CDK-dependent phosphorylation. Science. 271, 1597-1601 42 Hunter, T. and Plowman, G. D. (1997) The protein kinases of budding yeast: six score and more. Trends Biochem Sci. 22, 18-22 43 Kaouass, M., Audette, M., Ramotar, D., Verma, S., De Montigny, D., Gamache, I., Torossian, K. and Poulin, R. (1997) The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae. Mol Cell Biol. 17, 2994-3004 44 Mulet, J. M., Leube, M. P., Kron, S. J., Rios, G., Fink, G. R. and Serrano, R. (1999) A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter. Mol Cell Biol. 19, 3328-3337 45 Perez-Valle, J., Jenkins, H., Merchan, S., Montiel, V., Ramos, J., Sharma, S., Serrano, R. and Yenush, L. (2007) Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters. Mol Cell Biol. 27, 5725-5736 46 Roberg, K. J., Bickel, S., Rowley, N. and Kaiser, C. A. (1997) Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8. Genetics. 147, 1569-1584

13

Experiment 1 Purpose: Learn the basic steps involved in digesting a protein and

subsequent LC/MS/MS analysis. The model protein used is GST-Npr1 that

had been isolated from untreated or rapamycin-treated yeast cells.

Buffers, enzymes and solvents used

1. GST-Npr1, untreated and rapamycin treated

2. Solid urea

3. 100 mM DTT

4. 100 mM Tris-HCl, pH 8.0/6 M urea

5. 0.5 M iodoacetamide in 100 mM Tris-HCl, pH 8.0

6. Endoproteinase LysC (Achromobacter protease, Wako Chemicals), 1.0

µg/µl

7. 100 mM Tris-HCl, pH 8.0

8. Trypsin (sequencing grade, Promega), 0.25 µg/µl

9. Methanol

10. 80% acetonitrile/0.1% TFA

11. 0.1% TFA/1% acetonitrile

12. 0.4% TFA/4% acetonitrile

13. 0.1% acetic acid/2% acetonitrile

Procedure

Reduction, alkylation and digestion of GST-Npr1 is described for only one

sample (for example GST-Npr1 from untreated cells, but the GST-Npr1 from

rapamycin treated cells must be treated identically). Also, one half of the

digest is used for Experiment 1, the other half for Experiment 3 (see outline

below).

14

Fig. 1: Outline of the Experiments 1 and 3.

15

1. To a 100 µl aliquot of GST-Npr1 from untreated cells, add 57.6 mg

solid urea, vortex the solution until all urea dissolved and add 16 µl 100

mM DTT. The final volume will be around 160 µl and the urea

concentration will be about 6M.

2. Do the same for a 100 µl aliquot of GST-Npr1 from rapamycin treated

cells.

3. Incubate the solutions at 37oC for 1 hr to reduce the cysteines.

4. Add 20 µl 100 mM Tris-HCl, pH 8.0 in 6 M urea and 20 µl 0.5 M

iodoacetamide in 100 mM Tris-HCl, pH 8.0. Alkylate the cysteines at

room temperature for 15 min in the dark.

5. Add 0.50 µg endoproteinase LysC and incubate the samples for 1 hr at

37oC on a Thermoshaker. Cover the shaker with aluminium foil.

6. Dilute the digests by adding 400 µl 100 mM Tris-HCl, pH 8.0 to lower

the urea concentration to 2 M final. Add 0.5 µg trypsin, mix and

incubate at 37oC for 1 hr. Add a second 0.5 µg trypsin aliquot, mix and

incubate another 2 hrs at 37oC. Cover the shaker with aluminium foil.

7. In the meantime, prepare four MacroSpin Columns:

a. Place the MacroSpin columns into 2 ml Eppendorf tubes

b. Prime the columns: add 300 µl methanol on top of each column

packing and place the MacroSpin columns inserted into the

Eppendorf tubes into a centrifuge. Spin the columns at 800 rpm

for 2 min. Discard the methanol in the Eppendorf tubes.

c. Prime the MacroSpin columns with 300 µl 80% acetonitrile/0.1%

TFA as in steb 7b.

d. Equilibrate each MacroSpin column twice with 300 µl 0.1%

TFA/1% acetonitrile. Discard the liquid in the Eppendorf tubes.

8. Add 200 µl 0.4% TFA/4% acetonitrile to your digests. The TFA lowers

the pH such that trypsin will be inactivated. Spin the digests for 5 min at

10,000 rpm at room temperature. The final volume of your digests

(minus rapamycin/plus rapamycin) is 800 µl.

16

9. Distribute the digest of GST-Npr1 from untreated cells equally onto two

MacroSpin columns (400 µl onto each column) and do the same with

the GST-Npr1 from rapamycin treated cells (400 µl onto each column)

and place the columns into a fresh Eppendorf tubes.

10. Spin the columns at 800 rpm for 2 min and collect the flow throughs.

11. Reapply the flow throughs onto the columns and spin again at 800 rpm

for 2 min to ensure maximal peptide binding.

12. Wash the columns five times with 300 µl 0.1% TFA/1% acetonitrile to

remove the last traces of urea.

13. Place the MacroSpin columns into a fresh 2 ml Eppendorf tubes (label

them properly). Pipet 300 µl 80% acetonitrile/0.1% TFA onto each

individual column and elute bound peptides by spinning the columns

for 2 min at 800 rpm. At this stage, you have four eluates. Label one

eluate of the GST-Npr1 digest from non treated cells as A- and the

other eluate as B-. Label one eluate of the GST-Npr1 digest from

rapamycin treated cells as A+ and the other B+ (see Fig. 1).

14. Dry the A- and the A+ eluates separately in a speed vac. This takes

about 1 hr. Keep the B- and B+ eluates as they are used in

Experiment 3. Do not dry them. 15. Dissolve the A- and A+ digests each in 50 µl 0.1% acetic acid/2%

acetonitrile and spin at 10,000 rpm for 5 min.

16. Analyse the A- and A+ digests by LC/MS/MS. This will be done

together with the assistants.

17. Once we have a representative sample available, we will thoroughly

analyse the recorded spectra, as well as the protein identification either

by SEQUEST or PHENYX. Very importantly, we will try to look for

phosphopeptides as well as their location within the protein.

17

Experiment 2 Purpose: In-gel digestions to exploit the stability of Npr1 following rapamycin

treatment of yeast cells.

Buffers, enzymes and solvents used

1. 30% acrylamide

2. LGB (lower gel buffer) 4x: 1.5 M Tris-HCl, pH 8.8, 0.4% SDS

3. UGB (upper gel buffer) 4x: 0.5 M Tris-HCl, pH 6.8, 0.4% SDS

4. 10% ammonium sulfate (APS)

5. TEMED

6. 6x sample buffer

7. Running buffer 5x: 125 mM Tris-HCl, 0.96 M glycine, 0.5% SDS

8. Low-molecular weight standards

9. GST-Npr1, untreated and rapamycin treated

10. Bio-Safe Coomassie

11. SilverQuest kit

12. Ethanol

13. Acetic acid

14. Fixative: 40% ethanol/10% acetic acid

15. Wash: 30% ethanol

16. Sensitizer: 30% ethanol/10% Sensitizer from kit

17. Stainer: 1% stainer from kit

18. Developer: 10% Developer, to 100 ml solution, add 1 drop of

Developer enhancer

19. Stopper: to 100 ml Developer solution add 10 ml Stopper from kit

20. 40% n-propanol

21. 0.1M NH4HCO3/50% acetonitrile

22. 50 mM NH4HCO3

23. Trypsin (0.25 µg/µl in dissolution buffer)

24. 80% acetonitrile/0.1% acetic acid

25. 0.1% acetic acid/2% acetonitrile

18

Procedure

1. Assemble a cassette sandwich for a mini gel. Per cassette sandwich place a Short Plate on top of the Spacer Plate. Slide the two glass plates into the Casting Frame, keeping the Short Plate facing the front of the frame (side with pressure cams, see figure below).

2. When the glass plates are in place, lock the pressure cams to secure the glass cassette sandwich in the Casting Frame. Check that both plates are flush at the bottom. Secure the Casting Frame in the Casting Stand by engaging the spring-loaded lever (see figure below).

3. Place a comb into the assembled gel cassette. Mark the glass plate 1 cm below the comb teeth. This is the level to which the resolving gel is poured. Remove the comb.

4. Prepare a separating and stacking gel by combining all solutions listed in the table below except the TEMED.

Stock solutions Separating gel Stacking gel

30% acrylamide 3.00 ml 0.53 ml

LGB 4x 2.25 ml

UGB 4x 1.00 ml

Water 3.75 ml 2.47 ml

10% APS 45 µl 40 µl

TEMED 4.5 µl 4.0 µl

19

5. Add the TEMED to the separating gel to start polymerization. Pour the solution to the mark. Overlay the resolving gel immediately with water. Add the water evenly and smoothly to prevent mixing with the resolving gel solution. Let the gel polymerize completely.

6. When the Separating gel is completely polymerized, pour off the water from the surface. Add the TEMED to the stacking gel to start polymerization. Pour on top of the separating gel the stacking gel until the top of the Short Plate is reached. Insert the sample comb between the spacers. Seat the comb in the gel cassette by aligning the comb ridge with the top of the Short Plate.

7. When the stacking gel is completely polymerized, remove the Gel Cassette Assemblies from the Casting Stand. Rotate the cams to release the Gel Cassette Sandwich and place the Cassette Sandwich into the Electrode Assembly with the Short Plate facing inward. Slide Gel Cassette Sandwiches and Electrode Assembly into the clamping frame. Press down on the Electrode Assembly while closing the two cam levers of the Clamping Frame (see figures below). You will need a second gel cassette from another group that you put at the back of the Electrode Assembly to form a tightly sealed Inner Chamber.

8. Lower the Inner Chamber into the Mini Tank. Fill the Inner Chamber with 1 x running buffer till the level reaches halfway between the tops of the taller and shorter glass plates of the gel cassettes. Add about 200 ml 1 x running buffer to the Mini Tank. Put sample loading guides onto each of the gel sandwiches (see figure below).

20

9. Prepare the Low Molecular Weight Standard (LMW) by adding 10 µl of LMW diluted 1:20 into water, 20 µl water and 6 µl 6x sample buffer. Heat the sample to 95oC for 5 min.

10. To each of 30 µl GST-Npr1 from non treated and rapamycin treated cells add 6 µl 6x sample buffer and boil for 5 min at 95oC. Let the samples cool to room temperature and give it a quick spin to centrifuge moisture from the lid.

11. Load 15 µl each of the LMW, GST-Npr1 from untreated and rapamycin treated cells onto the gel according to the scheme below. Fill empty slots with sample buffer 1x (SB 1x).

12. Run the gel at 75V. When the Bromophenol blue front reaches the

separating gel, increase the voltage to 150V. 13. When the tracking dye reaches the bottom of the gel, switch off the

power supply and disassemble the cassette sandwich. 14. Put the gel onto a glass plate and cut it in two halves. One half is used

for staining with colloidal, the other half will be stained with silver.

21

Colloidal Blue Staining

1. Put one half of the gel into a staining tray and wash the gel with water three times for 5 min each. Use Nanopure water for the subsequent steps. Then add enough Bio-Safe Coomassie to just immerse the gel in staining solution. Stain the gel for 1 hr with gentle agitation on a rocking platform. This is a colloidal stain that does not fix the protein into the gel matrix so that peptides can be digested directly from the gel piece.

2. Pour off the stain and wash the gel three times with water for 5 min each. Destain the gel with water (Nanopure) till the background staining disappears and the bands become clearly visible.

3. Take a picture of your gel. Once you took a picture proceed with the steps outlined in the section Exploiting the rapamycin response of Npr1 by proteomics tools (see below).

Silver Staining

1. Immerse the gel in Nanopure water and wash it for 2 min on a rocking platform.

2. Fix the gel in 100 ml Fixative for 20 min (if time does not permit to carry out the entire staining protocol, fix the gel over night and continue the procedure the next day).

3. Decant the Fixative solution. Wash the gel in 100 ml 30% ethanol for 10 min.

4. Decant the 30% ethanol solution. Incubate the gel in 100 ml Sensitizing solution for 10 min.

5. Decant the Sensitizing solution. Wash the gel in 100 ml 30% ethanol for 10 min.

6. Wash the gel in 100 ml Nanopure water for 10 min. 7. Incubate the gel in 100 ml Staining solution for 15 min. 8. After staining, decant the Staining solution and wash the gel briefly

(for 20-60 seconds) in 100 ml Nanopure water. Washing the gel for more than a minute may remove silver ions from the gel resulting in decreased sensitivity.

9. Incubate the gel in 100 ml Developing solution for 4-8 min until the desired staining intensity is reached.

10. Stop the developing process by adding 10 ml Stopper directly to the gel still immersed in the Developing solution. Put the gel for 10 min onto the rocking platform and gently agitate the gel. The colour change from pink to colourless indicates that the development has stopped.

11. Decant the Stopper and wash the gel for 10 min with 100 ml Nanopure water.

12. Take a picture of your gel.

22

Exploiting the rapamycin response of Npr1 by proteomics tools

1. Place the gel that had been stained with Colloidal Blue onto a methanol-cleaned glass plate.

2. With a razor blade, cut the lanes of the Coomassie Blue stained gel BELOW the GST-Npr1 protein band from untreated and rapamycin treated cells into equal, horizontal gel slices of about 2 x 5 mm (see figure below). The silver stained gel will only be used as a reference as MS data from silver-stained bands are often poor. Each group should excise about four slices, so that the slices cover the entire gel from just below GST-Npr1 to the Coomassie Blue front. Put the gel pieces into pre-numbered Eppendorf tubes.

3. Carry out the in-gel digestions as described below. 4. Take the protein band out of the Eppendorf tube and put it on a clean

glass plate (wetted with a small aliquot of 40% n-propanol). 5. With a razor blade, cut the gel slice into small cubes. 6. With a spatula, transfer the gel pieces into an Eppendorf tube

containing 100 µl 40% n-propanol. Let the gel pieces settle to the bottom of the tube.

7. Wash the gel pieces five times with 100 µl 0.1 M NH4HCO3/50% acetonitrile. For each washing step, let the gel pieces settle by gravity and after they settled completely, aspirate the supernatant off and discard.

8. After the 5th wash, discard the supernatant and dry the gel pieces in a speed vac for approximately five minutes.

9. Dilute the trypsin stock solution 1:20 into 50 mM NH4HCO3. To the dried gel pieces, add 10 µl diluted trypsin.

10. Let the gel pieces swell so that the protease is completely taken up by the gel pieces. If the gel pieces are not completely immersed, add sufficient buffer (50 mM NH4HCO3) to completely immerse them.

11. Incubate the gel pieces over night at 37oC.

23

12. The next morning: add an equal volume 80% acetonitrile containing 0.1% acetic acid and incubate the gel pieces for 15 minutes at room temperature. This leads to shrinking of the gel pieces for easy collection of the supernatant.

13. Collect the supernatant (be careful not to remove any gel pieces) and pipet it into a new Eppendorf tube.

14. Dry the supernatant in a speed vac. 15. Dissolve the dried peptides in 50 µl 0.1% acetic acid containing 2%

acetonitrile. 16. Analyse the peptides by LC/MS/MS.

24

Experiment 3 Purpose: Relative quantification of changes of phosphorylation in Npr1

following rapamycin treatment.

Fig. 2: Outline of Experiment 3.

25

Buffers, enzymes and solvents used

1. TFA

2. Methanol

3. 80% acetonitrile/2.5% TFA

4. 0.1% TFA

5. 100 mM Na2HPO4, pH 8.9

6. 10% TFA

7. 0.1% acetic acid/2% acetonitrile

8. Dissolution buffer iTRAQ

9. Ethanol

10. 114/117-iTRAQ labels

11. Acetonitrile

12. 80% acetonitrile/0.1% TFA

1. To each of the B- and B+ eluates of the MacroSpin columns from

Experiment 1 add TFA to 2.5% final concentration (see Fig. 1 of

Experiment 1). Caution: TFA is harmful and may cause skin burns.

2. For phosphopeptide enrichment, prepare two TiO2 columns.

3. Prime each TiO2 tip twice with 200 µl water.

4. Prime each TiO2 tip twice with 200 µl methanol.

5. Equilibrate the tips three times with 200 µl 80% acetonitrile containing

2.5% TFA.

6. Load the B- and B+ digests onto the equilibrated TiO2 tips. Collect the

flow throughs.

7. Reapply the flow throughs onto the TiO2 tips. Repeat this five times to

bind as many phosphopeptides as possible.

8. Wash the tips six times with 200 µl 80% acetonitrile/2.5% TFA.

9. Wash the tips four times with 200 µl 0.1% TFA.

10. Elute the bound phosphopeptides from the TiO2 tips twice with 150 µl

100 mM Na2HPO4, pH 8.9.

26

11. Combine the first and second eluate of the B- digest and acidify with 30

µl 10% TFA. Combine the first and second eluate of the B+ digest and

acidify with 30 µl 10% TFA.

12. Desalt the B- and B+ eluates on separate MacroSpin columns as

described in Experiment 1.

13. Divide the desalted B- and B+ TiO2 phosphopeptides in two halves.

Dry one half of the desalted B- and B+ phosphopeptides in the speed

vac and redissolve in 50 µl 0.1% acetic acid/2% acetonitrile and

analyse them by LC/MS/MS (see Fig. 2).

14. Dry the other halves of the desalted TiO2 phosphopeptides in a

SpeedVac.

27

iTRAQ labelling of the phosphopeptide pools from untreated and rapamycin treated GST-Npr1

15. To 200 µl of the dissolution buffer of the iTRAQ kit (500 mM TEAB pH

8.5) add 200 µl Nanopure water and mix well.

16. Preparation of the iTRAQ labels (will be done by the instructors): warm

each iTRAQ reagent to room temperature and give it a quick spin. Add

140 µl dry ethanol, vortex and quick spin the reagents.

17. Dissolve your dried phosphopetides B- and B+ each in 6 µl diluted

dissolution buffer.

18. Add 14 µl iTRAQ label 114 to the B- phosphopeptides and 14 µl iTRAQ

label 117 to the B+ phosphopeptides. Vortex and give it a quick spin.

19. Incubate the phosphopeptides for 1 hr at room temperature.

20. Quench the reactions with 100 µl Nanopure water and incubate the

reaction mixtures at room temperature for another 30 min. Pool the

114- and 117-labelled peptides.

21. Dry the derivatised phosphopeptides in a speed vac.

22. Prepare ZipTips for desalting the iTRAQ’ed phosphopeptides.

23. Wet a ZipTip with 20 µl 100% acetonitrile.

24. Equilibrate the ZipTip three times with 20 µl 0.1% TFA.

25. Dissolve the dried iTRAQ-derivatised phosphopeptide pool in 36 µl

0.1% TFA and add 4 µl 10% TFA to quench any residual buffer. Spin

the solution at 10,000 rpm for 5 min.

26. Adsorb 20 µl of the derivatised phosphopeptides onto the ZipTips by

pipetting the solution up and down.

27. Wash the ZipTip five times with 20 µl 0.1% TFA.

28. Desorb the labelled phosphopeptides with 20 µl 80% acetonitrile/0.1%

TFA. Pipet the solution up and down a few times to bring about

complete desorption of bound peptides.

29. Dry the desalted iTRAQ’ed phosphopeptides in the speed vac.

30. Dissolve the phosphopeptides in 30 µl 0.1% acetic acid/2% acetonitrile

and centrifuge at 10,000 rpm for 10 min.

31. Analyse the peptides by LC/MS/MS.