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

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 γ 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. 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 γ 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. The regulation of cell surface receptors is essential for the maintenance of cell homeostasis and intercellular communication. At the plasma membrane ubiquitination has emerged as a critical post-translational mechanism for regulating expression of a wide range of surface proteins, including receptors of the immune system (1Staub O. Rotin D. Role of ubiquitylation in cellular membrane transport.Physiol. Rev. 2006; 86: 669-707Crossref PubMed Scopus (182) Google Scholar, 2Liu Y.C. Ubiquitin ligases and the immune response.Annu. Rev. Immunol. 2004; 22: 81-127Crossref PubMed Scopus (245) Google Scholar). The plasma membrane of immune cells hosts housekeeping receptors such as amino acid and ion transporters as well as a diverse range of proteins tailored to immune function. These include receptors for cellular and soluble ligands, antigen-presenting molecules, and adhesion molecules as well as cell-specific receptors such as NK 1The abbreviations used are:NKnatural killerCIITAMHC class II transactivator encoded by the gene AIR-1 (activator of immune response locus 1)CDcluster of differentiationEGFRepidermal growth factor receptorFcγRFc γ receptorGFPgreen fluorescent proteinHLAhuman leukocyte antigenICAMintercellular adhesion moleculeIgimmunoglobulinMARCHmembrane-associated RING-CHMHCmajor histocompatibility complexPTPRprotein-tyrosine phosphatase, receptor typePVRpolio virus receptorRINGreally interesting new geneRING-CHvariant RING domain, structurally relatedSAPSLAM-associated protein, also named SH2D1ASH3BP1Abl-SH3 domain-binding proteinSILACstable isotope labeling by amino acids in cell cultureSLAMsignaling lymphocytic activation molecule, also CD150VAMPvesicle-associated membrane proteinE1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseAPCallophycocyaninBis-Tris2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diolABCammonium bicarbonateLTQlinear trap quadrupoleHAhemagglutininEndo Hendoglycosidase HsiRNAshort interfering RNALIRleukocyte immunoglobulin-like receptorERendoplasmic reticulumESCRTendosomal sorting complex required for transportHheavyLlight. cell, T cell, and B cell receptor complexes. 350 cluster of differentiation (CD) molecules have been defined by monoclonal antibodies raised against cell surface proteins, and many of these are exclusive to lymphocytes (3Zola H. Swart B. Banham A. Barry S. Beare A. Bensussan A. Boumsell L. Buckley C.D. Bühring H.J. Clark G. Engel P. Fox D. Jin B.Q. Macardle P.J. Malavasi F. Mason D. Stockinger H. Yang X. CD molecules 2006–human cell differentiation molecules.J. Immunol. Methods. 2007; 319: 1-5Crossref PubMed Scopus (103) Google Scholar). The prominent role of transmembrane proteins in cellular function is emphasized by the observation that ∼20% of the genome codes for proteins with at least one hydrophobic α helix (4Arkin I.T. Brunger A.T. Statistical analysis of predicted transmembrane alpha-helices.Biochim. Biophys. Acta. 1998; 1429: 113-128Crossref PubMed Scopus (164) Google Scholar). natural killer MHC class II transactivator encoded by the gene AIR-1 (activator of immune response locus 1) cluster of differentiation epidermal growth factor receptor Fc γ receptor green fluorescent protein human leukocyte antigen intercellular adhesion molecule immunoglobulin membrane-associated RING-CH major histocompatibility complex protein-tyrosine phosphatase, receptor type polio virus receptor really interesting new gene variant RING domain, structurally related SLAM-associated protein, also named SH2D1A Abl-SH3 domain-binding protein stable isotope labeling by amino acids in cell culture signaling lymphocytic activation molecule, also CD150 vesicle-associated membrane protein ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase allophycocyanin 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol ammonium bicarbonate linear trap quadrupole hemagglutinin endoglycosidase H short interfering RNA leukocyte immunoglobulin-like receptor endoplasmic reticulum endosomal sorting complex required for transport heavy light. 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