title: Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells.
abstract: The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).
authors: Department of Molecular Pharmacology, The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors: Federica
authors: F
authors: Sotgia
authors: Babak
authors: B
authors: Razani
authors: Gloria
authors: G
authors: Bonuccelli
authors: William
authors: W
authors: Schubert
authors: Michela
authors: M
authors: Battista
authors: Hyangkyu
authors: H
authors: Lee
authors: Franco
authors: F
authors: Capozza
authors: Ann Lane
authors: AL
authors: Schubert
authors: Carlo
authors: C
authors: Minetti
authors: J Thomas
authors: JT
authors: Buckley
authors: Michael P
authors: MP
authors: Lisanti
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D000081247:CSK Tyrosine-Protein Kinase; D051242:Caveolin 1; D022461:Caveolins / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D002451:Cell Compartmentation; D006023:Glycoproteins / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism; D007684:Kidney Tubules / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014162:Transfection; D019061:src-Family Kinases
chemical_list: C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D019308:Palmitic Acid; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases