Apical localization of PMCA2w/b is lipid raft-dependent.
Alternative splicing of the first intracellular loop differentially targets plasma membrane calcium ATPase (PMCA) isoform 2 to the apical or basolateral membrane in MDCK cells. To determine if the targeting is affected by lipid interactions, we stably expressed PMCA2w/b and PMCA2z/b in MDCK cells, and analyzed the PMCA distribution by confocal fluorescence microscopy and membrane fractionation. PMCA2w/b showed clear apical and lateral distribution, whereas PMCA2z/b was mainly localized to the basolateral membrane. A significant fraction of PMCA2w/b partitioned into low-density membranes associated with lipid rafts. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced lipid raft association and a striking loss of PMCA2w/b from the apical membrane, whereas the lateral localization of PMCA2z/b remained unchanged. Our data indicate that alternative splicing differentially affects the lipid interactions of PMCA2w/b and PMCA2z/b and that the apical localization of PMCA2w/b is lipid raft-dependent and sensitive to cholesterol depletion.
Xiong
Yuning
Y
Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Antalffy
Géza
G
Enyedi
Agnes
A
Strehler
Emanuel E
EE
D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D004285:Dogs; D006801:Humans; D021962:Membrane Microdomains / Q000201:enzymology*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*
D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human