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This Article Free via Author's Choice: AC AC Full Text Full Text (PDF) Supplemental Data AC All Versions of this Article: M900174-MCP200v1 8/8/1959    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hör, S. Articles by Lehner, P. J. Search for Related Content PubMed PubMed Citation Articles by Hör, S. Articles by Lehner, P. J. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:1959-1971, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Stable Isotope Labeling by Amino Acids in Cell Culture and Differential Plasma Membrane Proteome Quantitation Identify New Substrates for the MARCH9 Transmembrane E3 Ligase^*,

Simon Hör, Tamar Ziv, Arie Admon and Paul J. Lehner^,¶

From the Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom and
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel

The regulation of cell surface receptor expression is essential for immune cell differentiation and function. At the plasma membrane ubiquitination is an important post-translational mechanism for regulating expression of a wide range of surface proteins. MARCH9, a member of the RING-CH family of transmembrane E3 ubiquitin ligases, down-regulates CD4, major histocompatibility complex-I (MHC), and ICAM-1 in lymphoid cells. To identify novel MARCH9 substrates, we used high throughput flow cytometry and quantitative mass spectrometry by stable isotope labeling by amino acids in cell culture (SILAC) to determine the differential expression of plasma membrane proteins in a MARCH9-expressing B cell line. This combined approach identified 13 potential new MARCH9 targets. All of the SILAC-identified targets for which antibodies were available were subsequently confirmed by flow cytometry, validating the proteomics results. A close correlation (r² = 0.93) between -fold down-regulation as determined by SILAC and flow cytometry was found, with no false positive hits detected. The potential new MARCH9 substrates cover a wide range of functions and include receptor-type protein-tyrosine phosphatases (e.g. PTPRJ/CD148) as well as Fc receptor IIB (CD32B), HLA-DQ, signaling lymphocytic activation molecule (CD150), and polio virus receptor (CD155). The identification of plasma membrane targets by SILAC with confirmation by flow cytometry represents a novel and powerful approach to analyze changes in the plasma membrane proteome.

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