Stable isotope labeling by amino acids in cell culture and differential plasma membrane proteome quantitation identify new substrates for the MARCH9 transmembrane E3 ligase

Abstract

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 (r(2) = 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 gamma 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.

Fig. 1.

Flow cytometry-based screen to identify novel MARCH9 substrates. A, Sultan MARCH9 B cells were generated by lentiviral transduction and co-expressed GFP from a separate promoter. The black line in the histogram represents the population gated for high expression of GFP, i.e. MARCH9-expressing; the filled gray line is the GFP-negative population; background levels are marked by the vertical dotted line that indicates the position of the peak of the control sample. B, surface expression of a panel of 44 B cell markers was compared between MARCH9-expressing cells and the untransduced GFP-negative population. The -fold reduction in mean fluorescence intensity is given for those samples that were repeatedly down-regulated. mIgD, membrane IgD.

Fig. 2.

Stable isotope labeling and plasma membrane preparation. A, Sultan B cells stably expressing MARCH9 were grown in medium only containing heavy arginine and lysine. After seven cell divisions equal cell numbers were pooled with control Sultan B cells grown in normal medium. The plasma membranes were purified with the pellicle method of Chaney and Jacobson (20), and the membrane proteins were eluted with SDS loading buffer. The protein mixture was separated on a precast SDS gel, divided into 10 pieces, tryptically digested, and analyzed by LC-MS/MS with an Orbitrap mass spectrometer. B, relative expression of all identified proteins plotted as a -fold change. Negative values indicate a down-regulation in the presence of MARCH9. The cutoff for a 2-fold change is indicated by the dotted line. The ratio of all proteins identified was 1:1.02 with a standard deviation of 0.31. wt, wild type.

Fig. 3.

Confirmation of mass spectrometry-identified candidates by flow cytometry. A and B, the change in cell surface expression of novel MARCH9 targets as identified by mass spectrometry was tested by flow cytometry. MARCH9-transduced Sultan B cells (generated as in Fig. 1) were stained for surface expression of the indicated markers. The -fold down-regulation is shown within each dot plot. B, the change in MHC class II expression in Sultan B cells was compared between MARCH9- (upper panel) and MARCH1 (lower panel)-transduced Sultan B cells. C, alignment of the transmembrane region and cytoplasmic tails of MHC class II (HLA-DQ, -DR, and -DP) chains. The transmembrane regions (underlined) and the cytoplasmic tails of class II α and β chains were aligned. Amino acids that are identical to those in HLA-DQ are shaded.

Fig. 4.

The -fold down-regulation of novel targets identified by SILAC MS correlates with flow cytometry. The -fold down-regulation of all targets identified by both SILAC MS and flow cytometry is compared. Regression analysis shows a linear correlation with a correlation coefficient (r²) of 0.93. The data points of the proteins displaying a change in expression levels are annotated. FACS, fluorescence-activated cell sorting.

Fig. 5.

Mechanism of substrate down-regulation. A, FcγRIIB or SLAM were co-transfected together with GFP-tagged wild-type or tryptophan to alanine mutant (W/A) MARCH9 into 293T cells and stained with antibodies specific for FcγRIIB or SLAM. The percentage of cells in each quadrant is shown. B, pulse-chase analysis of metabolically labeled HA-tagged SLAM in the presence or absence of MARCH9. HA-tagged SLAM-expressing 293T cells in the presence or absence of MARCH9 were [³⁵S]methionine-radiolabeled for 20 min and chased for the times indicated. Triton X-100 lysates were immunoprecipitated with HA-specific monoclonal antibody and treated with Endo H. Samples were analyzed by SDS-PAGE and autoradiography. The half-life (t½) of the Endo H-sensitive, immature form and the Endo H-resistant, mature form of SLAM is shown. C, Tsg101 siRNA-depleted 293T cells were co-transfected with SLAM and GFP-tagged MARCH9. SLAM and MARCH9 expression were analyzed by flow cytometry and compared with mock-depleted cells or cells transfected with tryptophan to alanine mutant MARCH9 (W/A). IP, immunoprecipitation.