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Submitted on April 25, 2007
Revised on July 12, 2007
Accepted on July 17, 2007
Dana-Farber Cancer Institute, Rm D1430, Boston, MA 02115
Corresponding Author: Martin_Hemler{at}DFCI.Harvard.EDU
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 have used a novel technical approach, involving exposure and crosslinking of membrane-proximal cysteines, coupled with LC-MS/MS protein identification. In this manner we have identified nine potential tetraspanin CD9 partners, including claudin-1. Chemical crosslinking 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 (-2, -3, -4, -5, -7) associated to a much lesser extent. Moreover, claudin-1 distributes very similarly to CD9 in sucrose gradients and, like CD9, is 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 (TJs), CD9–claudin-1 complexes did not reside in TJs, and depletion of key tetraspanins (CD9 and CD151) by siRNA 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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