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Contributions of quantitative proteomics to understanding membrane microdomains.

ABSTRACT: Membrane microdomains, e.g., lipid rafts and caveolae, are crucial cell surface organelles responsible for many cellular signaling and communication events, which makes the characterization of their proteomes both interesting and valuable. They are large cellular complexes comprised of specific proteins and lipids, yet they are simple enough in composition to be amenable to modern LC/MS/MS methods for proteomics. However, the proteomic characterization of membrane microdomains by traditional qualitative mass spectrometry is insufficient for distinguishing true components of the microdomains from copurifying contaminants or for evaluating dynamic changes in the proteome compositions. In this review, we discuss the contributions quantitative proteomics has made to our understanding of the biology of membrane microdomains.

SUBMITTER: Zheng YZ

PROVIDER: S-EPMC2739763 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Contributions of quantitative proteomics to understanding membrane microdomains.

Zheng Yu Zi YZ Foster Leonard J LJ

Journal of lipid research 20090703 10

Membrane microdomains, e.g., lipid rafts and caveolae, are crucial cell surface organelles responsible for many cellular signaling and communication events, which makes the characterization of their proteomes both interesting and valuable. They are large cellular complexes comprised of specific proteins and lipids, yet they are simple enough in composition to be amenable to modern LC/MS/MS methods for proteomics. However, the proteomic characterization of membrane microdomains by traditional qua ...[more]

PMID: 19578161

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Project description:Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma-associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif-deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins.

Dataset Information

Contributions of quantitative proteomics to understanding membrane microdomains.

Publications

Contributions of quantitative proteomics to understanding membrane microdomains.

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