Membrane raft actin deficiency and altered Ca2+-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis.
In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin-Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca2+-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin-actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.
Wilkinson
D Katie
DK
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Turner
E Jane
EJ
Parkin
Edward T
ET
Garner
Ashley E
AE
Harrison
Penny J
PJ
Crawford
Mark
M
Stewart
Gordon W
GW
Hooper
Nigel M
NM
D000199:Actins / Q000172:deficiency* / Q000378:metabolism; D000743:Anemia, Hemolytic / Q000378:metabolism*; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D005089:Exocytosis / Q000187:drug effects*; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051348:Tropomodulin / Q000378:metabolism
D000199:Actins; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D003902:Detergents; D008565:Membrane Proteins; C036860:STOM protein, human; D051348:Tropomodulin; D002118:Calcium