Split‐Ubiquitin System for Identifying Protein‐Protein Interactions in Membrane and Full‐Length Proteins

Christopher Grefen1, Sylvie Lalonde2, Petr Obrdlik3

1 Universität Tübingen, Tübingen, Germany, 2 Carnegie Institution, Stanford, California, 3 IonGate Biosciences GmbH, Frankfurt, Germany
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.27
DOI:  10.1002/0471142301.ns0527s41
Online Posting Date:  October, 2007
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Abstract

Protein-protein interactions play a fundamental role in the regulation of almost all cellular processes. Thus, the identification of interacting proteins can help to elucidate their function. The mating-based split-ubiquitin system (mbSUS) uses yeast as a test organism to identify potential interactions between full-length membrane proteins or between a full-length membrane protein and a soluble protein. The mbSUS can also be used to provide further evidence for protein-protein interactions detected with other methods and to map the interaction domains of selected proteins. The mbSUS is optimized for systematic screening approaches employing a mating-based approach, as typically used to determine protein interactions on a genomic scale. Construction of bait and prey fusions is simplified by adapting two different cloning procedures: (i) in vivo cloning in yeast, and (ii) Gateway cloning in E. coli. Protocols for small-scale interaction tests, as well as systematic approaches using sorted bait and prey arrays, are described. Curr. Protoc. Neurosci. 41:5.27.1-5.27.41. © 2007 by John Wiley & Sons, Inc.

Keywords: Split-ubiquitin; mbSUS; protein-protein interaction; full-length membrane protein; mating; systematic analysis; Gateway

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cloning of Cub-PLV and Nub Fusion Constructs
  • Basic Protocol 2: Characterization of Mating-Based Split-Ubiquitin System Bait Fusions
  • Basic Protocol 3: Systematic Split-Ubiquitin Interaction Tests with Selected Bait and Prey
  • Alternate Protocol 1: Cloning of Mating-Based Split-Ubiquitin Systems Fusions in E. coli
  • Alternate Protocol 2: Systematic Screening of Sorted Collections of Split-Ubiquitin Bait and Prey Fusions
  • Alternate Protocol 3: Quantitative Analysis of Interactions Between Cub-PLV and Nub Fusions
  • Support Protocol 1: Use of Different Mating-Based Split-Ubiquitin System Nub Vectors for Interaction Tests
  • Support Protocol 2: Plasmid Rescue from Yeast: “Lazy Bones” Protocol
  • Support Protocol 3: Preparation of Protein Extracts for Immunoblot Analysis
  • Support Protocol 4: X-Gal Overlay Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cloning of Cub-PLV and Nub Fusion Constructs

 Materials
  • DNA templates encoding bait and prey proteins of interest
  • mbSUS-gate vectors of choice (Table 5.27.2)
  • Restriction enzymes (appendix 1M) appropriate for cutting mbSUS-gate vector
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MAT URA3 leu2 trp1 his3 loxP::ade2)
  • YPAD liquid medium: YPD medium (see recipe) supplemented with 2 mg/liter adenine sulfate
  • LiAc/TE buffer (see recipe)
  • Sheared salmon sperm DNA (unit 4.4)
  • 1× TE buffer (see recipe for 10×)
  • 1 M lithium acetate (LiAc; see recipe)
  • PEG/LiAc mix (see recipe)
  • Synthetic-complete (SC) medium plates (see recipe):
    • SC/–Leu plates for transformation of THY.AP4 with Cub constructs
    • SC/–Trp plates for transformation of THY.AP5 with Nub constructs
  • Synthetic-complete (SC) liquid medium (see recipe) containing 200 µg/ml (final concentration) G418:
    • SC/–Leu + 200 µg/ml G418 for THY.AP4 bait CubPLV clones
    • SC/–Trp + 200 µg/ml G418 for clones of THY.AP5 prey
  • Synthetic-complete (SC) liquid medium (see recipe) without G418
    • SC/–Leu for THY.AP4 bait CubPLV clones
    • SC/–Trp for clones of THY.AP5 prey
  • Zymoprep II Yeast Plasmid Miniprep Kit (Zymo Research)
  • LB medium (appendix 2A) containing 100 µg/ml ampicillin
  • 28°C shaking incubator
  • Centrifuge
  • 30° and 42°C shaking water baths
  • Additional reagents and equipment for PCR amplification of DNA (cpmb unit 15.1), agarose gel electrophoresis (appendix 1N), isolation of DNA from agarose gels and purification of DNA via affinity columns (cpmb unit 2.6), transformation of bacteria by electroporation (appendix 1E), miniprep isolation of bacterial DNA (appendix 1J), and DNA sequencing (CPMB Chapter 7)

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 2: Characterization of Mating-Based Split-Ubiquitin System Bait Fusions

 Materials
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MAT URA3 leu2 trp1 his3 loxP::ade2)
  • DNA vectors pNX-gate32-3HA (Fig. 5.27.6A) and pNubWT-Xgate expressing soluble NubG and NubWT peptides, respectively (Table 5.27.2)
  • DNA constructs encoding bait-Cub-PLV fusions of interest (Basic Protocol 1)
  • Synthetic complete liquid medium (see recipe):
    • SC/–Leu
    • SC/–Trp
  • Synthetic-complete (SC) medium plates (see recipe) supplemented with l-methionine (Met) for repression of bait levels:
    • SC/–Leu plates
    • SC/–Trp plates
    • SC/–Trp, –Leu, –Ura, –Met, supplemented with different concentrations of Met
    • SC/–Trp, –Leu, –Ura, –Ade, –His, –Met, supplemented with different concentrations of Met
    • YPD medium and plates (see recipe)
    • 28°C shaking incubator
    • Centrifuge
  • Additional reagents and equipment for lithium acetate transformation (Basic Protocol 1; also see cpmb unit 13.7), replica plating of yeast (cpmb unit 13.2), and X-Gal overlay assay (Support Protocol 4)

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

Basic Protocol 3: Systematic Split-Ubiquitin Interaction Tests with Selected Bait and Prey

 Materials
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MAT URA3 leu2 trp1 his3 loxP::ade2)
  • DNA constructs encoding bait-Cub-PLV fusions (Basic Protocol 1), which passed the quality control in Basic Protocol 2
  • NubG-prey or prey-NubG fusions of interest (Basic Protocol 1)
  • DNA vectors pNXgate32-3HA, NubWT-Xgate and pMetYCgate (Table 5.27.2)
  • Synthetic-complete (SC) medium plates (see recipe; standard size plates as well as 12 × 12 cm square plates) supplemented with methionine (Met) for repression of bait levels
    • SC/–Leu plates
    • SC/–Trp plates
    • SC/–Trp, –Leu, –Ura, –Met, supplemented with different concentrations of Met
    • SC/–Trp, –Leu, –Ura, –Ade, –His, –Met, supplemented with different concentrations of Met
  • YPD plates (see recipe), square, 12 × 12 cm
  • 28°C incubator
  • Camera or flat-bed scanner for documenting successful mating on plates
  • Additional reagents and equipment for lithium acetate transformation (Basic Protocol 1; also see cpmb unit 13.7), mating and selection of bait and prey fusions (Basic Protocol 2), replica plating of yeast (cpmb unit 13.2), and X-Gal overlay assay (Support Protocol 4)

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and proper aseptic technique should be used accordingly.

Alternate Protocol 2: Systematic Screening of Sorted Collections of Split-Ubiquitin Bait and Prey Fusions

 Materials
  • DNA templates for bait and prey (Basic Protocol 1)
  • pMetYCgate and Nub vectors of choice (see Support Protocol 1 and Commentary) for in vivo cloning of bait and prey B1-ORF-B2 PCR products (see Basic Protocol 1 for more detail)
  • mbSUS-gate vectors pMetYCgate, pNXgate32-3HA and pNubWT-Xgate as controls (Table 5.27.2)
  • Yeast strains (Table 5.27.1)
    • THY.AP4 (MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2)
    • THY.AP5 (MAT URA3 leu2 trp1 his3 loxP::ade2)
  • Synthetic-complete (SC) medium plates (see recipe; standard size plates as well as 12 × 12 cm square plates):
    • SC/–Leu plates
    • SC/–Leu plates + 200 µg/ml G418
    • SC/–Trp plates
    • SC/–Trp plates + 200 µg/ml G418
  • 45% (v/v) glycerol, sterile
  • YPD plates (see recipe), square, 12 × 12 cm
  • Synthetic-complete (SC) liquid medium:
    • SC/–Leu
    • SC/–Trp
    • SC/–Trp, –Leu, –Ura, –Ade, –His, –Met, supplemented with different concentrations of Met
  • 24-well plates
  • 96-well microtiter plates
  • 96-pin replicator: Multi-Blot VP407 (V&P Scientific) or Replication System (Nalge Nunc)
  • Spectrophotometer with microtiter plate reader
  • Additional reagents and equipment for cloning of Cub-PLV and Nub fusion constructs (Basic Protocol 1)

NOTE: All solutions and materials coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

Alternate Protocol 3: Quantitative Analysis of Interactions Between Cub-PLV and Nub Fusions

 Materials
  • SC/–Trp, –Leu, –Ura microtiter plate arrays with diploid cells (Basic Protocol 3, step 7)
  • SC/–Trp, –Leu, –Ura liquid medium (see recipe)
  • Z buffer (see recipe), ice cold
  • Liquid nitrogen
  • Acid-washed glass beads (see recipe)
  • Bradford reagent (see recipe)
  • Protein standard: 1 mg/ml BSA in Z buffer
  • ortho-nitrophenyl-galactopyranoside (o-NPG)
  • 1 M Na2CO3
  • 28°C shaking incubator
  • Refrigerated centrifuge
  • Spectrophotometer
  • Heating block
  • Additional reagents and equipment for preparing microtiter plate arrays of bait and prey (Basic Protocol 3)

Support Protocol 2: Plasmid Rescue from Yeast: “Lazy Bones” Protocol

 Additional Materials (also see Basic Protocol 1)
  • THY.AP4 and THY.AP5 clones (see Basic Protocol 1)
  • Acid-washed glass beads (see recipe)
  • DNA release solution (see recipe)
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol
  • Isopropanol
  • 80% (v/v) ethanol
  • Additional reagents and equipment for transformation of E. coli (appendix 1E and cpmb unit 1.8)

Support Protocol 3: Preparation of Protein Extracts for Immunoblot Analysis

 Materials
  • THY.AP4 and THY.AP5 clones (Basic Protocol 1) or diploid yeast clones (Basic Protocol 3)
  • Synthetic-complete (SC) liquid medium (see recipe):
    • SC/–Leu, –Trp, –Ura
    • SC/–Leu
    • SC/–Trp
  • Lysis buffer: 0.2 M NaOH/0.2% (v/v) 2-mercaptoethanol (prepare fresh)
  • 100% trichloroacetic acid (see recipe)
  • 0.5 M Tris×Cl, pH 6.8 (appendix 2A)
  • Anti-VP16 (Abcam, http://www.abcam.com) and anti-HA antibodies (Sigma-Aldrich)
  • 28°C incubator
  • Refrigerated centrifuge
  • Additional reagents for SDS-PAGE and immunoblotting (unit 5.19)

Support Protocol 4: X-Gal Overlay Assay

 Additional Materials (also see Basic Protocols 2 and 3)
  • Diploid cells expressing different bait and prey fusions (Basic Protocol 2 and 3)
  • Z buffer (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS) in H2O
  • 100 mg/ml X-Gal stock solution in dimethylformamide
  • 50°C water bath
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Figures

  •  FigureFigure 5.27.1 Flow chart of different strategies for testing protein-protein interactions with mbSUS.
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  •  FigureFigure 5.27.2 The mbSUS for detection of protein-protein interactions. Cub-PLV peptide is gray, NubG peptide is black. (A) Bait and prey proteins do not interact. (B) Interaction between bait and prey proteins enables reconstitution of functional ubiquitin. The functional ubiquitin is recognized by ubiquitin specific proteases (USPs), which release the artificial transcription factor “PLV” (ProteinA-LexA-VP16; shown as gray oval). The released PLV binds lexA-regulated promoters (white boxes) in the nucleus and activates the reporter genes ADE2, HIS3, and lacZ. The activation of ADE2 and HIS3 can be detected by complementing the growth of yeast cells on medium lacking adenine and histidine. The activation of lacZ can be visualized via qualitative and quantitative -galactosidase assays. To enable the cleavage of PLV by USPs, NubG, and Cub peptides must be on the cytosolic face of the membrane.
    View Image
  •  FigureFigure 5.27.3 Schematic model of the cloning cassettes of different mbSUSgate and mbSUS-DEST vectors. Promoters are depicted as white, coding sequences as dark gray arrows. The B1/B2 linkers are light grey, the Gateway attachment sites attR1/R2 are black arrows. (A) The cloning cassette of mbSUS-gate vectors was designed for recombinational cloning in yeast. The black triangle in the cloning cassette of pXNgate21-3HA symbolizes the “mini-ORF” ATGTGA reducing the expression level of the cloned fusion protein (see also Fig. 5.27.7). (B) Cloning cassette of mbSUS-DEST Destination vectors suitable for Gateway cloning of mbSUS fusions in E. coli.
    View Image
  •  FigureFigure 5.27.4 Flow chart of the cloning strategies for mbSUS constructs. The chart demonstrates that an identical mbSUS construct can be cloned via two different cloning strategies. The “ORF” box indicates the open reading frame of the selected gene of interest. (A) PCR strategy for cloning an ORF into mbSUS vectors. The B1 forward primer consists of 36 bp, equal to the B1 linker, followed by the ATG and 20 to 25 additional nucleotides specific to the 5¢-region of the ORF. The B2 reverse primer consists of 39 nucleotides complementary to the B2 linker and 20 to 25 nucleotides—excluding a stop codon—specific to the 3¢-end of the chosen ORF. The inset of the arrows flanking the ORF shows the sequence of the linker-specific regions of the forward and reverse primers. Gateway attachment sites attB1 and attB2 in the primer sequence are bold and underlined. (B) Construction of mbSUS fusions via recombinational in vivo cloning in yeast. (C) Construction of mbSUS fusions via Gateway cloning in E. coli. Gray arrows indicate the B1 and B2 linkers and the complementary attP sites of a pDONR vector. White arrows indicate the attL and attR sites of the entry clone and the destination vector, respectively.
    View Image
  •  FigureFigure 5.27.5 Maps of mbSUS Cub-PLV vectors. Black arrows symbolize the selection markers as well as the KanMX and Gateway cloning cassettes including the B1/B2 linkers and the attR1/attR2 sites, respectively. The detailed structure of both cloning cassettes is described in Figure 5.27.3. White arrows indicate the Met25 promoter region for the expression of bait-CubPLV fusions. Dark gray arrows with white letters indicate the Cub-PLV tag. Light gray boxes symbolize the different origins of replication for maintenance E. coli and for yeast. The vector pMetYCgate (A) and the vector pMetYC-DEST (B) differ only in the cloning cassette designed either for the in vivo cloning approach in yeast or for Gateway cloning, respectively. The Met25 promoter allows regulation of bait expression with methionine. Both vectors are low-copy ARS/CEN plasmids in yeast.
    View Image
  •  FigureFigure 5.27.6 Maps of mbSUS-gate vectors for N-terminal NubG-prey fusions. Black arrows symbolize the selection markers as well as the KanMX cloning cassette including B1 and B2 linkers. The detailed structure of the cloning cassette is described in Figure 5.27.3. White arrows indicate the ADH promoter region for the expression of NubG-prey fusions. Dark gray arrows indicate the NubG and HA-tags. Light gray boxes symbolize the different origins of replication. In yeast, the vector pNXgate32-3HA (A) is a high-copy plasmid containing a 2µ origin of replication and suited for high expression of NubG-prey fusions. pNXgate33-3HA (B) is a low-copy ARS/CEN plasmid for low expression of NubG-prey constructs.
    View Image
  •  FigureFigure 5.27.7 Maps of mbSUS-gate vectors for C-terminal prey-NubG fusions. The symbols and colors are as described in Figure 5.27.6. In yeast, both vectors are high-copy plasmids due to the 2µ origin of replication. (A) The map of the vector pXNgate21-3HA. The blowup section shows sequence detail of the post-promoter region of pXNgate21-3HA. The “mini-ORF” ATGTGA reduces the efficiency of the expression and hence keeps the levels of the fusion protein low in spite of the high copy number of the plasmid. (B) The map of the vector pXNgate22-HA. The start codon of the mini-ORF is mutated in the pXNgate22-HA vector thus allowing higher expression levels of prey in comparison to pXNgate21-3HA. Note that pXNgate22-HA bears only a single HA tag.
    View Image
  •  FigureFigure 5.27.8 Maps of mbSUS-DEST destination vectors for constructing prey fusions via Gateway cloning. The symbols and colors are as described in Figures 5.27.5 and 5.27.6. Both vectors are high-copy plasmids designed for high expression of prey fusions in yeast. The vector pNX32-DEST (A) is suited for N-terminal NubG-prey fusions, whereas pXN22-DEST (B) is designed for constructing C-terminal prey-NubG fusions.
    View Image
  •  FigureFigure 5.27.9 An example of a cell array on four square petri dishes designed for systematic small-to-medium scale interaction tests (Basic Protocol 3). 13 different THY.AP4 clones harboring 12 different bait fusions and the vector pMetYCgate were mixed with 13 different THY.AP5 clones harboring 11 different prey fusions and the vectors pNXgate32-3HA and pNubWT-Xgate. 4-µl drops of cell mixes were arrayed in four square petri dishes containing YPD. The THY.AP4 clones are numbered, whereas THY.AP5 clones are labeled with letters. The clones carrying the vectors are highlighted. The array was propagated by replica plating as described in Basic Protocol 3.
    View Image

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Literature Cited

Literature Cited
    Dirnberger, D., Unsin, G., Schlenker, S., and Reichel, C. 2006. A small-molecule–protein interaction system with split-ubiquitin as sensor. ChemBioChem 7:936-942.
    Gietz, R.D., Schiestl, R.H., Willems, A.R., and Woods, R.A. 1995. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355-360.
    Griffith, D.A., Delipala, C., Leadsham, J., Jarvis, S.M., and Oesterhelt, D. 2003. A novel yeast expression system for the overproduction of quality-controlled membrane proteins. FEBS Lett. 553:45-50.
    Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. 2000. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. U.S.A. 98:4569-4574.
    Johnsson, N. and Varshavsky, A. 1994. Split-ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. U.S.A. 91:10340-10344.
    Kaiser, C., Michaelis, S., and Mitchell, A. 1994. Methods in Yeast Genetics, a Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York.
    Matsuda, S., Giliberto, L., Matsuda, Y., Davies, P., McGowan, E., Pickford, F., Ghiso, J., Franqione, B., and D'Adamio, L. 2005. The familial dementia BRI2 gene binds the Alzheimer gene amyloid-beta precursor protein and inhibits amyloid-beta production. J. Biol. Chem. 280:28912-28916.
    Obrdlik, P., El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarinoe, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent, V., Blaudez, D., Sanders, D., Revuelta, J.L., Boles, E., Andre, B., and Frommer, W.B. 2004. K+ channel interactions detected by a genetic system optimized for systematic studies of membrane protein interactions. Proc. Natl. Acad. Sci. U.S.A. 101:12242-12247.
    Osborne, A.R., Rapoport, T.A., and van den Berg, B. 2005. Protein translocation by the Sec61/SecY channel. Annu. Rev. Cell Dev. Biol. 21:529-550.
    Pandey, S. and Assmann, S.M. 2004. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling. Plant Cell 16:1616-1632.
    Raquet, X., Eckert, J.H., Müller, S., and Johnsson, N. 2001. Detection of altered protein conformations in living cells. J. Mol. Biol. 305:927-938.
    Reinders, A., Schulze, W., Kühn, C., Barker, L., Schulz, A., Ward, J.M., and Frommer, W.B. 2002. Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split ubiquitin system and functional assays. Plant Cell 14:1567-1577.
    Rubio-Somoza, I., Martinez, M., Abraham, Z., Diaz, I., and Carbonero, P. 2006. Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds. Plant J. 47:269-281.
    Schwacke, R., Schneider, A., van der Graaff, E., Fischer, K., Catoni, E., Desimone, M., Frommer, W.B., Flügge, U.I., and Kunze, R. 2003. ARAMEMNON: A novel database for Arabidopsis integral membrane proteins. Plant Physiol. 131:16-26.
    Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. 1998. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. U.S.A. 95:5187-5192.
    Supply, P., Wach, A., Thinès-Sempoux, D., and Goffeau, A. 1993. Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae. J. Biol. Chem. 268:19744-19752.
    Uetz, P., Giot, L., Cagney, G., Mansfield, T.A., Judson, R.S., Knight, J.R., Lockshon, D., Narayan, V., Srinivasan, M., Pochrat, P., Quereshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J.M. 2000. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403:623-627.
    Ugolini, S. and Bruschi, C.V. 1996. The red/white colony color assays in the yeast Saccharomyces cerevisiae: Epistatic growth advantage of white ade-8-18, ade2 cells over red ade2 cells. Curr. Genet. 30:485-492.
    Wang, B., Pelletier, J., Massaad, M.J., Herscovics, A., and Shore, G.C. 2004. The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B. Mol. Cell Biol. 24:2767-2778.
    Wittke, S., Lewke, N., Müller, S., and Johnsson, N. 1999. Probing the molecular environment of membrane proteins in vivo. Mol. Biol. Cell 10:2519-2530.
    Zhang, J. and Lautar, S. 1996. A yeast three-hybrid method to clone ternary protein complex components. Anal. Biochem. 242:68-72.
 Key References
    Johnsson and Varshavsky, 1994. See above.

Initial description of the split-ubiquitin.

    Obrdlik et al., 2004. See above.

Initial description of mbSUS. The paper explains the features of mbSUS and introduces several methods described in this unit.

 Internet Resources
    http://www.invitrogen.com

Commercial resources for Gateway Entry plasmids and for Gateway cloning reagents.

    http://www.biosci.ohio-state.edu/pcmb/Facilities/abrc/abrchome.htm

The ABRC database is the source for mbSUS yeast strains as well as the majority of mbSUS vectors.

    http://www.associomics.org/goodies/split_ubiquitin/index.html

Overview of all split ubiquitin components available at the ABRC database and their ABRC catalog numbers.

    http://www.cbs.dtu.dk/services/TMHMM/

TMHMM Server v. 2.0. Web server for prediction of transmembrane helices in proteins.

    http://www.enzim.hu/hmmtop/index.html

HMMTOP: Program for prediction of transmembrane helices and topology of proteins.

    http://www.cbs.dtu.dk/services/SignalP/

SignalP 3.0 server: Predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms.

    http://www.expasy.ch/tools/#topology

Tools for topology and signal peptide prediction.

    http://aramemnon.botanik.uni-koeln.de

Aramemnon database, providing a comparative view of the topology of plant membrane proteins.

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