A Novel Cysteine Cross-linking Method Reveals a Direct Association between Claudin-1 and Tetraspanin CD9 Export

@article{citeulike:3467106, 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. 10.1074/mcp.M700183-MCP200}, author = {Kovalenko, Oleg V. and Yang, Xiuwei H. and Hemler, Martin E.}, citeulike-article-id = {3467106}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/mcp.M700183-MCP200}, citeulike-linkout-1 = {http://www.mcponline.org/cgi/content/abstract/6/11/1855}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/17644758}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=17644758}, day = {1}, doi = {10.1074/mcp.M700183-MCP200}, journal = {Mol Cell Proteomics}, keywords = {association, cd9, cross-linking, cysteine, identification, interaction, method, screen, tetraspanin}, month = {November}, number = {11}, pages = {1855--1867}, posted-at = {2008-10-30 23:38:45}, priority = {2}, title = {A Novel Cysteine Cross-linking Method Reveals a Direct Association between Claudin-1 and Tetraspanin CD9}, url = {http://dx.doi.org/10.1074/mcp.M700183-MCP200}, volume = {6}, year = {2007} }

TY - JOUR ID - citeulike:3467106 L3 - citeulike-article-id:3467106 N2 - 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. 10.1074/mcp.M700183-MCP200 IS - 11 JF - Mol Cell Proteomics EP - 1867 TI - A Novel Cysteine Cross-linking Method Reveals a Direct Association between Claudin-1 and Tetraspanin CD9 VL - 6 SP - 1855 KW - association KW - cd9 KW - cross-linking KW - cysteine KW - identification KW - interaction KW - method KW - screen KW - tetraspanin AU - Kovalenko, Oleg AU - Yang, Xiuwei AU - Hemler, Martin PY - 2007/11/01/ UR - http://dx.doi.org/10.1074/mcp.M700183-MCP200 ER -