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Translocation of caveolin into the endoplasmic reticulum and transport of cholesterol to caveolae requires a functional “flip domain”
Author(s) -
Guerin Theresa,
Smart Eric J
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1180.5
Subject(s) - caveolae , caveolin , endoplasmic reticulum , caveolin 1 , microbiology and biotechnology , cytosol , biology , cytoplasm , caveolin 3 , chemistry , biochemistry , signal transduction , enzyme
We previously demonstrated that a caveolin‐chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. We also showed that the depletion of caveolae with cholesterol oxidase induces caveolin to translocate to the lumen of the ER. We hypothesized that the amino acid sequence, VxKxWF, functions as a “flip domain” to facilitate translocation of caveolin into the ER. To test this hypothesis, tryptophan‐98 was changed to phenylalanine. A myc‐tag was added to the C‐terminal ends of wild type caveolin (wt‐myc) and the mutant caveolin (flip‐myc) and both constructs were stably expressed in Swiss 3T3 cells. Both wt‐myc and flip‐myc caveolin localized to caveolae as determined by subcellular fractionation and indirect immunofluorescence. Treatment of cells with cholesterol oxidase caused wt‐myc caveolin to translocate from caveolae to the ER and flip‐myc caveolin to translocate from the caveolae to the cytosol. When wt‐myc and flip‐myc were expressed in L1210 cells, both proteins formed a complete caveolin‐chaperone but only wt‐myc rapidly transported newly synthesized cholesterol from the ER to caveolae. We conclude that tryptophan‐98 in the putative caveolin “flip domain” is necessary for the translocation of caveolin into the ER and for the rapid transport of cholesterol to caveolae.