Evaluation of Prototype Transmembrane 4 Superfamily Protein Complexes and Their Relation to Lipid Rafts

Abstract

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 ␣ 3 ␤ 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 ␣ 3 integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-␣3␤1 and CD81-␣3␤1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-␣3␤1, CD81-␣3␤1, 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°C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-␣3␤1, CD81-␣3␤1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.

Caveolin-1 Regulates Matrix Metalloproteinases-1 Induction and CD147/EMMPRIN Cell Surface Clustering

“…Complexes containing caveolin-1 typically appear in the lowdensity "light membrane" fractions of sucrose gradients (38). Indeed, from Brij 96 lysates of A431 cells, the bulk of caveolin-1 appeared in fractions (lanes 1-5) containing low density (5-35%) sucrose (Fig.…”

“…A431 cells (1-2 ϫ 10 6 ) were lysed in 2 ml of Mes-buffered saline (MBS; 25 mM Mes, pH 6.5, 0.15 M NaCl) containing 1% Brij 96 and proteinase inhibitor for 2-4 h, passed through a 22-gauge needle to shear DNA, and then Dounce homogenized for 10 strokes. The homogenate was adjusted to 40% sucrose by addition of 80% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube, and then centrifuged at 45,000 rpm for 16 -20 h in a SW41 rotor in a discontinuous 5, 35, and 45% sucrose density gradient (38). 12 fractions of 400 l each were collected from the top of the gradient, and then CD147 complex was immunoprecipated from each fraction, using mAb 8G6.…”

Phosphoinositides in Constitutive Membrane Traffic

“…A PtdIns4KII activity coprecipitates with CD63, a tetraspannin protein found mainly on lysosomes (50,231), and is also found in plasma membrane lipid rafts, although it is not enriched in rafts that contain caveolin (376). The single yeast homolog of mammalian PtdIns4KIIs is LSB6, now renamed PIK2.…”

CDw78 Defines MHC Class II-Peptide Complexes That Require Ii Chain-Dependent Lysosomal Trafficking, Not Localization to a Specific Tetraspanin Membrane Microdomain

“…Palmitoylation of many proteins, including tyrosine kinases, leads to their recruitment into lipid raft microdomains (9,10). While tetraspanins are indeed palmitoylated, tetraspanins are thought to exist in detergent-insoluble, but distinct "nonraft" membrane microdomains, since the detergent-insolubility of tetraspanins is thought to be refractory to cholesterol depletion (11).…”