The annexin II2p11(2) complex is the major protein component of the triton X-100-insoluble low-density fraction prepared from MDCK cells in the presence of Ca2+.
Annexin II2p11(2) is present in the submembranous region of cells expressing both subunits of the complex. Most probably, this subcellular distribution is maintained through the interaction of annexin II2p11(2) with membrane phospholipids and/or elements of the cortical cytoskeleton known to occur in vitro in a Ca(2+)-dependent manner. To determine whether membrane or cytoskeleton interactions are primarily responsible for anchoring annexin II2p11(2) in the cell cortex, we subjected Madin-Darby canine kidney (MDCK) cells to serial extractions using different detergents and identified annexin II and p11 in the different fractions employing specific antibodies. The complex but not monomeric annexin II remains insoluble when the cells are extracted with Triton X-100 in the presence of Ca2+. However, treatment of the Triton X-100-insoluble cell remnants with a series of other detergents known to solubilize GPI-anchored proteins leads to a partial extraction of annexin II2p11(2) even in the presence of Ca2+. Using sucrose density gradient analysis in the presence of Ca2+ as a different means of fractionating the Triton X-100-insoluble cell remnants we show that the majority of annexin II2p11(2) copurifies with a low-density fraction which has been reported to contain GPI-anchored proteins, certain glycolipids, and VIP21/caveolin. Annexin II2p11(2) is by far the most abundant protein component in this fraction indicating that its association with the low-density material occurs via lipid binding and is not due to the interaction with a certain protein.
Harder
T
T
Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Gerke
V
V
D000818:Animals; D017306:Annexin A2 / Q000737:chemistry* / Q000302:isolation & purification; D002118:Calcium / Q000494:pharmacology*; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D017830:Octoxynol; D012995:Solubility; D013696:Temperature
D017306:Annexin A2; D017830:Octoxynol; D002118:Calcium