TY - JOUR
T1 - A novel cysteine cross-linking method reveals a direct association between claudin-1 and tetraspanin CD9
AU - Kovalenko, Oleg V.
AU - Yang, Xiuwei H.
AU - Hemler, Martin E.
PY - 2007/11
Y1 - 2007/11
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.
AB - 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.
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U2 - 10.1074/mcp.M700183-MCP200
DO - 10.1074/mcp.M700183-MCP200
M3 - Article
C2 - 17644758
AN - SCOPUS:36749037222
SN - 1535-9476
VL - 6
SP - 1855
EP - 1867
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 11
ER -