title: A novel cysteine cross-linking method reveals a direct association between claudin-1 and tetraspanin CD9.
abstract: Tetraspanins serve as molecular organizers of multiprotein microdomains in cell membranes. Hence to understand functions of tetraspanin proteins, it is critical to identify laterally interacting partner proteins. Here we used a novel technical approach involving exposure and cross-linking of membrane-proximal cysteines coupled with LC-MS/MS protein identification. In this manner we identified nine potential tetraspanin CD9 partners, including claudin-1. Chemical cross-linking yielded a CD9-claudin-1 heterodimer, thus confirming direct association and adding claudin-1 to the short list of proteins that can directly associate with CD9. Interaction of CD9 (and other tetraspanins) with claudin-1 was supported by subcellular colocalization and was confirmed in multiple cell lines, although other claudins (claudin-2, -3, -4, -5, and -7) associated to a much lesser extent. Moreover claudin-1 was distributed very similarly to CD9 in sucrose gradients and, like CD9, was released from A431 and A549 cells upon cholesterol depletion. These biochemical features of claudin-1 are characteristic of tetraspanin microdomain proteins. Although claudins are major structural components of intercellular tight junctions, CD9-claudin-1 complexes did not reside in tight junctions, and depletion of key tetraspanins (CD9 and CD151) by small interfering RNA had no effect on paracellular permeability. However, tetraspanin depletion did cause a marked decrease in the stability of newly synthesized claudin-1. In conclusion, these results (a) validate a technical approach that appears to be particularly well suited for identifying protein partners directly associated with tetraspanins or with other proteins that contain membrane-proximal cysteines and (b) provide insight into how non-junctional claudins may be regulated in the context of tetraspanin-enriched microdomains.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Kovalenko
authors.forename: Xiuwei H
authors.initials: XH
authors.lastname: Yang
authors.forename: Martin E
authors.initials: ME
authors.lastname: Hemler
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D062445:Claudin-1; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D060245:Tetraspanin 29
chemical_list: D015703:Antigens, CD; C554869:CD9 protein, human; C568108:CLDN1 protein, human; D062445:Claudin-1; D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D060245:Tetraspanin 29; D003545:Cysteine