Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts

Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha (3)beta (1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria, First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha (3) integrin colocalized with ganglioside GM1 as seen by double staining of Bred cells. Third, CD9-alpha3 beta1 and CD81-alpha3 beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3 beta1, CD81-alpha3 beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains, These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degreesC, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3 beta1, CD81-alpha3 beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.