A Genome Wide Screening Approach for Membrane-targeted Proteins*,S

Hanna Jaaro, Zehava Levy and Mike Fainzilber{ddagger}

From the Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.


Secreted and cell surface molecules are critical for intercellular communication, especially in the nervous system. Indeed given the vast diversity of cell types in the brain, unraveling the complexity of chemical communication in the central nervous system (CNS)1 poses a daunting challenge. In recent years it has become apparent that a single molecule or even a single molecular family focus on intercellular communication is of insufficient scope to lead to a broad understanding of system functions. Although genetic screens in model organisms can be performed for system wide analyses, genes with subtle loss-of-function phenotypes or pleiotropic roles are unlikely to be identified. Furthermore classical genetic screens do not provide a focus on specific functional molecular classes (e.g. secreted versus intracellular). Accordingly a number of groups have tried to develop approaches that would allow rapid and comprehensive identification of all secreted gene products (the "secretome") in a given cell or tissue. These include direct proteomic approaches utilizing mass spectrometry (14), library preparation from RNA enriched by microsomal fractionation (5, 6), and so-called signal sequence trap methods in yeast or mammalian cells (7, 8).

The signal sequence trap approach is conceptually attractive since it utilizes a functional selection or screening step to select all the cDNAs encoding secreted proteins in a given cell or tissue. All signal trap systems are based on reporter expression or selection to identify a gene product that is directed to the cell surface if fused to a foreign cDNA incorporating a secretion signal such as an N-terminal signal peptide. Signal trap methods have been used to characterize partial secretomes in systems ranging from cellular models of prostate cancer to extracellular communication in plants (912). Most available signal trap methods require lengthy expression and selection protocols in mammalian cells and are therefore both labor-intensive and time-consuming. To circumvent these limitations, a signal trap method was devised in a yeast strain mutated for the enzyme invertase and consequently not capable of growth on sucrose medium (8). Transfection of this mutant with a library of cDNA fragments fused to mature invertase rescued those clones that received cDNA fusions targeted for secretion. The approach provided the largest data set of secreted gene products from all studies to date (13). However, since the invertase must be secreted to metabolize sucrose, receptor fusions incorporating transmembrane stretches or peripheral proteins membrane-linked by lipid posttranslational modifications are not preferentially selected by this method.

Transmembrane and other membrane-associated proteins include receptors, ion channels, cell adhesion molecules, and transport/internalization modulators. These all play important roles in mediating cell-cell interactions, cell growth and differentiation, activation, and apoptosis in all cell types. Most of the "druggable targets" of current interest to the pharmaceutical industry fall into this category (4). Despite their undoubted importance in cellular physiology, biochemical purification and characterization of membrane-associated components is difficult, hampering progress in structural and functional characterization of membrane proteins. We therefore sought to develop a comprehensive membrane protein trapping method akin to the signal trap systems in yeast. To this end we modified the yeast protein interaction screen termed the Ras recruitment system (RRS, Ref. 14) to allow direct selection of membrane-targeted proteins. The modified system, termed the Ras membrane trap (RMT), was validated in a pilot screen on the CNS of the mollusk Lymnaea, revealing 112 membrane-associated proteins including two representatives of gene families so far not found in invertebrates.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Construction of a Lymnaea CNS cDNA Library for RMT—
150 Lymnaea CNS were dissected from adult snails as described previously (15). To obtain a comprehensive library the CNSs were divided into two batches, one of which was processed immediately for mRNA extraction, while the second batch underwent interganglionic crush followed by 2 days of incubation in vitro followed by mRNA extraction. The mRNAs were pooled before cDNA synthesis primed with random hexamers fused to a HindIII restriction site. The cDNA was then ligated with BamHI adaptors, digested with HindIII, and ligated into the pMET-Ras vector, which was constructed by transferring Ras from pADH-Ras to the pMET vector (16) as shown (see Fig. 1A). Transformation to electrocompetent bacteria yielded a primary library of 3 x 105 clones.



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FIG. 1. Ras membrane trap: overview and screen. A, principle of the method. Only inserts encoding membrane-targeted proteins will bring the fused Ras to the vicinity of the plasma membrane, thus allowing yeast growth. B, map of the pMet-Ras vector, indicating the library cloning sites. The Met25 promoter shuts down expression from the plasmid in the presence of methionine. C, schematic of the main stages of an RMT screen. Transformation and incubation at 24 °C is followed by replica-plating and selection at the restrictive temperature of 36 °C. Positive clones are then picked and replated for elimination of false positives by methionine selection. D, an example of methionine selection at the final step of a screen. Positive colonies are those that grow in the absence of methionine and are repressed by addition of 50 µg/ml methionine. E, categories of clones obtained from the Lymnaea CNS library. Left, membrane association of the selected clones as predicted by on-line software tools. Right, subcellular localization of close homologs for those clones with informative homologies in the data bases. PM, plasma membrane; ER, endoplasmic reticulum; mito, mitochondria; cytoskel, cytoskeleton; sec, secreted.

 
Screening in Saccharomyces cerevisiae—
The yeast S. cerevisiae cdc-25 strain was maintained and transformed as described previously (17). Transformants were plated on glucose minimal medium excluding uracil, which is the marker for selection by in pMet-Ras. After 5 days at 24 °C colonies were replica-plated and incubated at the selection temperature of 36 °C. The surviving colonies were screened for false positives by adding methionine (50 mg/liter, Sigma, M-2893) to shut down expression from the plasmid. Colonies that grew poorly in the presence of methionine at 36 °C were chosen for sequence analysis.

Bioinformatic Analyses—
On-line tools were used for prediction of membrane association as follows. Transmembrane protein prediction was carried out with TMHMM (18), a global approach based on a hidden Markov model to predict the most probable topology for the whole protein, and TMpred (19), a local approach based on statistical analysis of the TMbase data base. These tools were accessed at www.cbs.dtu.dk/services/TMHMM-2.0/ and www.ch.embnet.org/software/TMPRED_form.html, respectively. Myristoylation prediction was carried out on NMT (20) at mendel.imp.univie.ac.at/myristate/.

Transfection and Immunostaining in Mammalian Cells—
The cDNA insert and Ras were excised together from the pMet vector with BamHI and NotI and transferred to pCDNA3. The resulting plasmids were transfected to COS-7 cells by the DEAE-dextran method. Transfected cells were permeabilized and stained with anti-pan-Ras (Oncogene, OP40, 1:50) and anti-p75 intracellular domain (Covance, PRB-602C, 1:1000) followed by rhodamine red-X (Jackson ImmunoResearch, 715-295-151, 1:500)- and Cy5 (Jackson ImmunoResearch, 711-175-152, 1:500)-conjugated secondaries, respectively. Sections were observed under an Olympus FV500 confocal laser scanning microscope at x60 magnification. For rhodamine red-X and Cy5 visualization, we used 543 and 633 nm wavelengths in a sequential manner.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
We aimed to develop a comprehensive method for the identification of membrane-associated gene products, including both peripherally associated and integral membrane proteins, by modifying the RRS method for identifying protein-protein interactions. RRS is based on the strict requirement for plasma membrane localization of Ras for survival and growth of S. cerevisiae, normally achieved by farnesylation of the CAAX box of Ras (21). In the temperature sensitive cdc25-2 strain the endogenous Ras pathway is inactivated at 36 °C. This property is utilized for selection in RRS by rescuing clones co-transfected with two interacting proteins, a library clone that is usually targeted by myristoylation and a bait protein fused to a CAAX-deleted mammalian Ras. Mammalian Ras is localized at the membrane via a protein-protein interaction, enabling the transfected yeast to grow (14). A recent modification termed "reverse RRS" utilizes a membrane protein as bait and a library fused to Ras (22). We realized that the system can be used for direct assessment of membrane targeting of the Ras-fused constructs by dispensing with the interacting partner and relying on a methionine-sensitive repressor for clone verification (16). A schematic of the system, termed the RMT is shown in Fig. 1.

We first verified the system using constructs of the transmembrane with extracellular domain versus the intracellular domain alone of the mammalian p75 receptor. After obtaining the expected specific rescue of the transmembrane-containing clone only (data not shown), we carried out a pilot screen on a random primed cDNA library from the CNS of the mollusk Lymnaea, a model organism from a phylum that to date lacks a representative with a sequenced genome. The library was constructed by cloning cDNA inserts 5' to Ras, thus potentially allowing selection of type I transmembrane proteins, lipid-conjugated (myristoylated or palmitoylated) membrane proteins, or peripheral proteins brought to the membrane via non-covalent interactions. Library DNA was transfected into yeast, and 1.2 x 106 clones were plated on minimal glucose medium lacking uracil and methionine. Transformants were incubated for 4 days at 24 °C and then replica-plated and selected at the restrictive temperature of 36 °C. Approximately 2,700 transformants were picked from the 36 °C plates and rescreened with and without methionine (Fig. 1D). 516 colonies that passed this second round screen were processed, sequenced, and clustered, resulting in 112 unique sequences of which 109 are novel (GenBankTM accession numbers AY577317AY577426).

Homology analyses by Blast (23) revealed significant homologies for 32 of the novel sequences, seven of these from Lymnaea or Aplysia (Supplemental Table I). The subcellular localization of known homologs and membrane association predictions for all sequences are shown in Fig. 1E. TMHMM predicted only 28% of the selected sequences as membranal, whereas the less restrictive TMpred predicted 45% transmembrane proteins, including all those found by TMHMM. Only two sequences started with Met-Gly (which is an absolute requirement for myristoylation), and neither were predicted as a potential myristoylation substrate by NMT. Subcellular localization of the 32 known homologs turned out to be primarily on intracellular membranes with high representation for components of the endoplasmic reticulum or the nucleus (Fig. 1E). To verify membrane association of sequences lacking significant homologies in the data bases, we selected eight cDNAs not predicted as membranal by the software tools we used and subcloned them together with the fused Ras to the mammalian expression vector pCDNA3. We then co-transfected COS-7 cells with the resulting plasmids together with a membrane marker (p75) and co-stained the cells after permeabilization with pan-Ras and p75 antibodies. Co-localization of the two markers was observed for all the studied clones in some cases restricted specifically to plasma membrane and in others also observed in cytoplasmic vesicles, most likely components of the secretory or internalization pathways (Fig. 2).



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FIG. 2. Localization of selected clones in mammalian COS-7 cells. Clones lacking predicted membrane-targeting signals or known homologs were transferred to the pCDNA3 mammalian expression vector. The resulting plasmids were co-transfected to COS cells together with p75 as a plasma membrane marker. Cells were fixed after 36 h and immunostained for p75 (green) and Ras (red). Yellow indicates co-localization.

 
Interestingly two of the clones were found to have vertebrate but not Caenorhabditis elegans or Drosophila homologs. Clone AY577335 is homologous to a sequence identified in a large scale effort to identify novel human secreted and transmembrane proteins (13) and related to a Hedgehog-interacting protein (Fig. 3). Clone AY577329 is a member of the squalene epoxidase family, endoplasmic reticulum membrane proteins that are key enzymes in sterol biosynthesis. Squalene epoxidases are found from fungi to humans but were not identified in C. elegans or Drosophila genome sequences.



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FIG. 3. Phylogenetic trees of the closest eukaryotic homologs of clones AY577333 and AY577329. Trees were generated by the neighbor-joining method using default parameters of the ClustalX program. SE, squalene epoxidase. Note the absence of Drosophila or C. elegans homologs in both cases. Alignments of the sequences comprising these trees are shown in the supplemental material on line.

 
Our screen was limited by the size of the pilot library and the fact that only one-sixth of the inserts should be in the correct frame. Moreover a clone will be selected only if it contains both an ATG translation initiation codon and a membrane-targeting signal in-frame with Ras. The average size of library inserts was ~400 bp. If an average membrane protein is ~1,000 amino acids, one-eighth of the clones may include the membrane-targeting sequence, and we make a random assumption that only one-tenth of the clones containing membrane-targeting signals will also have an in-frame ATG. Given all these caveats and assumptions, we would predict 500–1,000 unique positives from screening a library of 300,000 independent clones. The fact that only ~100 sequences were obtained in the screen could be due to library quality issues or other technical problems or could be due to differences between mollusks and yeast in processing and identification of membrane-targeting motifs as was previously reported in a comparison of signal sequence utilization between yeast and mammals (24).

The software packages we used to analyze the results of the screen categorized only about half of the clones as membrane-targeted. Indeed a comparative evaluation of these and other transmembrane prediction programs showed that reliability of the analyses is tightly correlated with a tendency to underpredict (25). Nonetheless verification of a subset of the selected clones showed that they were indeed targeted to membranes upon transfection to mammalian cells (Fig. 2). As long as better in silico methods are lacking, comprehensive characterization of membrane proteomes will have to be driven by experimental approaches. RMT is a flexible method for genome wide analyses of membrane proteomes with the advantage that it can also cover proteomes of intracellular membranes. The advent of reverse RRS screening for protein-protein interactions using membrane proteins as baits and libraries fused to Ras (22) means that libraries appropriate for RMT are likely to become increasingly available. Since the membrane proteome encoded in such libraries represents "background noise" for the protein-protein interaction screens for which they were designed, RMT can extract interesting data from the discarded clones of a reverse RRS screen. Improvements in library design such as larger inserts, directional cloning, and inclusion of initiation codons in all three frames of the vector will make the method even more effective in the future.

Interestingly two of the clones obtained in the screen had significant homology to vertebrate genes, but no C. elegans or Drosophila homologs. How can evolution explain genes common to vertebrates and molluscs and not existing in C. elegans and Drosophila? Although classical morphology-based phylogeny tagged molluscs and arthropods as coelomates and nematodes as pseudocoelomates, certain molecular phylogenies place arthropods and nematodes on the same branch of the evolutionary tree (26, 27). Thus, the ancestral proteins that existed before the branch point between mammals and molluscs may have been lost in the common ancestor of nematodes and flies. This draws attention to the importance of expanding genome sequence projects to cover representatives of all major phyla.


    ACKNOWLEDGMENTS
 
We thank Eitan Reuveny and Ami Aronheim for generous gifts of reagents and advice.


    FOOTNOTES
 
Received, November 28, 2004, and in revised form, December 30, 2004.

Published, MCP Papers in Press, December 31, 2004, DOI 10.1074/mcp.T400020-MCP200

1 The abbreviations used are: CNS, central nervous system; NMT, N-terminal N-myristoylation prediction program; RMT, Ras membrane trap; RRS, Ras recruitment system; TMHMM, transmembrane helices prediction program; TMPred, transmembrane regions and orientation prediction program. Back

* This work was supported by funds from the Weizmann Institute Genome Center and the Abisch-Frenkel Foundation. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

The nucleotide sequences reported in this paper have been submitted to GenBankTM/EBI Data Bank with the accession numbers AY577317AY577426.

{ddagger} The incumbent of the Daniel E. Koshland Sr. Career Development Chair at the Weizmann Institute. To whom correspondence should be addressed. Tel.: 972-8-934-4266; Fax: 972-8-934-4112; E-mail: mike.fainzilber{at}weizmann.ac.il


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