Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

Federica Sotgia, Babak Razani, Gloria Bonuccelli, William Schubert, Michela Battista, Hyangkyu Lee, Franco Capozza, Ann Lane Schubert, Carlo Minetti, J. Thomas Buckley, Michael P. Lisanti

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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 ∼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).

Original languageEnglish
Pages (from-to)3905-3926
Number of pages22
JournalMolecular and Cellular Biology
Volume22
Issue number11
DOIs
Publication statusPublished - 2002 May 28

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Caveolins
Inositol
Caveolin 1
Caveolin 3
Proteins
Lipoylation
Null Lymphocytes
Caveolin 2
Caveolae
Lipids
Cell Membrane
Skeletal Muscle Fibers
Golgi Apparatus
Protein Transport
Cysteine
Membrane Proteins
Down-Regulation
Embryonic Structures
Fibroblasts

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Sotgia, Federica ; Razani, Babak ; Bonuccelli, Gloria ; Schubert, William ; Battista, Michela ; Lee, Hyangkyu ; Capozza, Franco ; Schubert, Ann Lane ; Minetti, Carlo ; Buckley, J. Thomas ; Lisanti, Michael P. / Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells. In: Molecular and Cellular Biology. 2002 ; Vol. 22, No. 11. pp. 3905-3926.
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Sotgia, F, Razani, B, Bonuccelli, G, Schubert, W, Battista, M, Lee, H, Capozza, F, Schubert, AL, Minetti, C, Buckley, JT & Lisanti, MP 2002, 'Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells', Molecular and Cellular Biology, vol. 22, no. 11, pp. 3905-3926. https://doi.org/10.1128/MCB.22.11.3905-3926.2002

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells. / Sotgia, Federica; Razani, Babak; Bonuccelli, Gloria; Schubert, William; Battista, Michela; Lee, Hyangkyu; Capozza, Franco; Schubert, Ann Lane; Minetti, Carlo; Buckley, J. Thomas; Lisanti, Michael P.

In: Molecular and Cellular Biology, Vol. 22, No. 11, 28.05.2002, p. 3905-3926.

Research output: Contribution to journalArticle

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T1 - Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

AU - Sotgia, Federica

AU - Razani, Babak

AU - Bonuccelli, Gloria

AU - Schubert, William

AU - Battista, Michela

AU - Lee, Hyangkyu

AU - Capozza, Franco

AU - Schubert, Ann Lane

AU - Minetti, Carlo

AU - Buckley, J. Thomas

AU - Lisanti, Michael P.

PY - 2002/5/28

Y1 - 2002/5/28

N2 - 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 ∼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).

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