Abstract
Membrane-associated and secreted proteins are an important class of proteins and include receptors, transporters, adhesion molecules, hormones and cytokines. Although algorithms have been developed to recognize potential amino-terminal membrane-targeting signals or transmembrane domains in protein sequences, their accuracy is limited and they require knowledge of the entire coding sequence, including the N terminus1, which is not currently available for most of the genes in most organisms, including human. Several experimental approaches for identifying secreted and membrane proteins have been described, but none have taken a comprehensive genomic approach2,3,4,5,6. Furthermore, none of these methods allow easy classification of clones from arrayed cDNA libraries, for which large-scale gene-expression data are now becoming available through the use of DNA microarrays. We describe here a rapid and efficient method for identifying genes that encode secreted or membrane proteins. mRNA species bound to membrane-associated polysomes were separated from other mRNAs by sedimentation equilibrium or sedimentation velocity. The distribution of individual transcripts in the ‘membrane-bound’ and ‘cytosolic’ fractions was quantitated for thousands of genes by hybridization to DNA microarrays. Transcripts known to encode secreted or membrane proteins were enriched in the membrane-bound fractions, whereas those known to encode cytoplasmic proteins were enriched in the fractions containing mRNAs associated with free and cytoplasmic ribosomes. On this basis, we identified over 275 human genes and 285 yeast genes that are likely to encode previously unrecognized secreted or membrane proteins.
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References
Nielsen, H., Brunak, S. & von Heijne, G. Machine learning approaches for the prediction of signal peptides and other protein sorting signals. Protein Eng. 12, 3–9 (1999).
Tashiro, K. et al. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261, 600–603 (1993).
Klein, R.D., Gu, Q., Goddard, A. & Rosenthal, A. Selection for genes encoding secreted proteins and receptors. Proc. Natl Acad. Sci. USA 93, 7108–7113 (1996).
Zannettino, A.C., Rayner, J.R., Ashman, L.K., Gonda, T.J. & Simmons, P.J. A powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning. J. Immunol. 156, 611–620 (1996).
Kopczynski, C.C. et al. A high throughput screen to identify secreted and transmembrane proteins involved in Drosophila embryogenesis. Proc. Natl Acad. Sci. USA 95, 9973–9978 (1998).
Scherer, P.E., Bickel, P.E., Kotler, M. & Lodish, H.F. Cloning of cell-specific secreted and surface proteins by subtractive antibody screening. Nature Biotechnol. 16, 581–586 (1998).
Mechler, B. & Rabbitts, T.H. Membrane-bound ribosomes of myeloma cells. IV. mRNA complexity of free and membrane-bound polysomes. J. Cell. Biol. 88, 29–36 (1981).
Mueckler, M.M. & Pitot, H.C. Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations. J. Cell. Biol. 90, 495–506 (1981).
Kyte, J. & Doolittle, R.F. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132 (1982).
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6 (1997).
Takizawa, P.A., Sil, A., Swedlow, J.R., Herskowitz, I. & Vale, R.D. Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast. Nature 389, 90–93 (1997).
Sidrauski, C. & Walter, P. The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell 90, 1031–1039 (1997).
Chapman, R.E. & Walter, P. Translational attenuation mediated by an mRNA intron. Curr. Biol. 7, 850–859 (1997).
Lukyanetz, E.A. Evidence for colocalization of calcineurin and calcium channels in dorsal root ganglion neurons. Neuroscience 78, 625–628 (1997).
Pietrini, G. et al. A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase. J. Cell. Biol. 117, 975–986 (1992).
Mechler, B.M. Isolation of messenger RNA from membrane-bound polysomes. Methods Enzymol. 152, 241–248 (1987).
Spellman, P.T. et al. Comprehensive identification of cell cycle-regulated genes of the yeast saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273–3297 (1998).
Sherman, F. Getting started with yeast. Methods Enzymol. 194, 3–21 (1991).
Stoltenburg, R., Wartmann, T., Kunze, I. & Kunze, G. Reliable method to prepare RNA from free and membrane-bound polysomes from different yeast species. Biotechniques 18, 564–566, 568 (1995).
Kacharmina, J.E., Crino, P.B. & Eberwine, J. Preparation of cDNA from single cells and subcellular regions. Methods Enzymol. 303, 3–18 (1999).
Eisen, M.B. & Brown, P.O. DNA arrays for analysis of gene expression. Methods Enzymol. 303, 179–205 (1999).
DeRisi, J.L., Iyer, V.R. & Brown, P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686 (1997).
Iyer, V.R. et al. The transcriptional program in the response of human fibroblasts to serum. Science 283, 83–87 (1999).
Perou, C.M. et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc. Natl Acad. Sci. USA 96, 9212–9217 (1999).
Egea, G., Izquierdo, J.M., Ricart, J., San Martin, C. & Cuezva, J.M. mRNA encoding the beta-subunit of the mitochondrial F1-ATPase complex is a localized mRNA in rat hepatocytes. Biochem. J. 322, 557–565 (1997).
Lightowlers, R.N., Sang, A.E., Preiss, T. & Chrzanowska-Lightowlers, Z.M. Targeting proteins to mitochondria: is there a role for mRNA localization? Biochem. Soc. Trans. 24, 527–531 (1996).
Acknowledgements
We thank the members of the Brown and Botstein labs for assistance and discussions, and M. Niwa, J. Peters and P. Walter for helpful advice and assistance. This work was supported by the Howard Hughes Medical Institute and by grants from the NHGRI (HG00983) and the NCI (CA77097). M.D. was supported by an MSTP fellowship. M.B.E. was supported by a DOE/NSF Sloan Fellowship. P.O.B. is an associate investigator of the Howard Hughes Medical Institute.
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Diehn, M., Eisen, M., Botstein, D. et al. Large-scale identification of secreted and membrane-associated gene products using DNA microarrays. Nat Genet 25, 58–62 (2000). https://doi.org/10.1038/75603
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DOI: https://doi.org/10.1038/75603
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