Caveolin and MAL, Two Protein Components of Internal Detergent-Insoluble Membranes, Are in Distinct Lipid Microenvironments in MDCK Cells

doi:10.1006/bbrc.1997.6530

Biochemical and Biophysical Research Communications

Volume 233, Issue 3, 28 April 1997, Pages 707-712

https://doi.org/10.1006/bbrc.1997.6530 Get rights and content

Abstract

The MAL proteolipid and caveolin have been identified as components of internal detergent-insoluble membrane microdomains enriched in glycolipids and cholesterol. We have addressed the study of the glycolipid-enriched membranes in cells expressing endogenously only either MAL (Jurkat T cells) or caveolin (epithelial A498 cells) and in polarized MDCK cells which express both proteins simultaneously. Subcellular fractionation by centrifugation to equilibrium in sucrose gradients of Triton X-100 cell extracts from Jurkat and A498 cells revealed that MAL and caveolin are incorporated in detergent-insoluble buoyant membranes independently of the expression of each other and indicated the existence in these cells of insoluble membrane microdomains with either MAL or caveolin. Immunofluorescence analysis in MDCK cells indicated that both MAL and caveolin were located in the Golgi region, whereas caveolin was found in addition at the cell surface. Biochemical analysis in these cells revealed the existence of distinct membrane microenvironments differentially susceptible to detergent solubilization containing either internal MAL or internal plus surface caveolin. The observed heterogeneity within the internal glycolipid-enriched membrane fraction suggests the existence of distinct specialized lipid microenvironments in MDCK cells.

References (22)

D.A. Brown et al.
Cell
(1992)
K. Simons et al.
Cell
(1990)
K.G. Rothberg et al.
Cell
(1992)
S. Li et al.
J. Biol. Chem.
(1996)
D. Zacchetti et al.
FEBS Lett.
(1995)
C. Rancaño et al.
J. Biol. Chem.
(1994)
P. Chomczynski et al.
Anal. Biochem.
(1987)
R.G. Parton
Curr. Opin. Cell Biol.
(1996)
E. Rodriguez-Boulan et al.
Science
(1989)
M.P. Lisanti et al.
J. Cell Biol.
(1989)

There are more references available in the full text version of this article.

Cited by (40)

Epsilon toxin from Clostridium perfringens induces cytotoxicity in FRT thyroid epithelial cells
2018, Anaerobe
Citation Excerpt :
MAL protein is expressed in FRT and in MDCK cells [26–29]. In addition, in MDCK cells, caveolin and MAL are two protein components of internal DRMs, although in distinct lipid microenvironments [39]. Here, we have described a new cell line of thyroid origin that does not express caveolin 1 but is sensitive to Etx and could be a useful model to study MAL-Etx interaction.
Epsilon toxin (Etx) is produced by Clostridium perfringens and induces enterotoxemia in ruminants. Etx crosses the blood-brain barrier, binds to myelin structures, and kills oligodendrocytes, inducing central nervous system demyelination. In addition, Etx has a cytotoxic effect on distal and collecting kidney tubules. There are few cell lines sensitive to Etx. At present, the most sensitive in vitro model for Etx is the Madin-Darby canine kidney (MDCK) cell line, where Etx oligomerizes and forms a pore with consequent ion efflux and cell death. Although the Etx receptor has not yet been fully clarified, it is known that caveolin 1 and 2 potentiate Etx cytotoxicity and oligomerization, and more recently, the myelin and lymphocyte (MAL) protein has been implicated in Etx binding and activity. Here, we studied the effect of Etx on Fischer rat thyroid cells (FRT) and observed similar effects as those seen in MDCK cells. Etx incubated with FRT cells showed binding to the plasma membrane, and western blotting assays revealed oligomeric complex formation. Moreover, cytotoxic assays on FRT cells after Etx incubation indicated cell death at a similar level as in MDCK cells. In addition, a luminescent ATP detection assay revealed ATP depletion in FRT cells after Etx exposure. Previous studies have reported that FRT cells do not express caveolins and do not form caveolae but express MAL protein in glycolipid-enriched membrane microdomains. Our results indicate that caveolins are not directly implicated in Etx cytotoxicity, supporting the notion that the MAL protein is involved in Etx action. In addition, a cell line of thyroid origin is described for the first time as a good model to study Etx action.
Productive entry of type C foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol
2007, Virology
Citation Excerpt :
The rabbit polyclonal antibody anti-caveolin 1 used showed that both cell lines expressed caveolin-1, being the level of expression detected higher in BHK-21 cells. As reported (Millan et al., 1997), no caveolin-1 expression was detected in Jurkat cells, used as control. Fig. 6B shows the immunofluorescence pattern of caveolin-1 in IBRS-2, which appeared as membrane-associated vesicles, corresponding to lipid-rafts and caveolae, as well as with a perinuclear distribution characteristic of a Golgi location, as reported (Kurzchalia et al., 1994).
We have characterized the entry leading to productive infection of a type C FMDV in two cell lines widely used for virus growth, BHK-21 and IBRS-2. Inhibition of clathrin-mediated endocytosis by sucrose treatment decreased both cell entry and virus multiplication. Evidence of a direct requirement of clathrin for productive viral entry was obtained using BHK21-tTA/anti-CHC cells, which showed a significant reduction of viral entry and infection when the synthesis and functionality of clathrin heavy chain was inhibited (Tet− cells). This was also observed for vesicular stomatitis virus (VSV) productive entry. The effect of NH₄Cl and concanamycin A on FMDV entry and infection was consistent with the requirement of acidic compartments for decapsidation and virus replication. As expected from its higher stability at acidic pH, this requirement was higher for VSV. Since BHK-21 and IBRS-2 cells expressed caveolin-1, we explored the effect on productive virus entry of drugs that interfere with caveolae-mediated endocytosis. Treatment with nystatin did not reduce entry and infection of FMDV or VSV, while cholesterol depletion with MβCD significantly inhibited both steps of the FMDV cycle, indicating that plasma membrane cholesterol is required for virus productive entry.
Fas ligand is localized to membrane rafts, where it displays increased cell death-inducing activity
2006, Blood
Citation Excerpt :
Despite the copurification of caveolin by Lalor et al,56 it remains likely that rafts had in fact been isolated together with the caveolae. Our own conclusion that FasL is localized to rafts rather than to caveolae (at least in those cells analyzed by us) is based on the fact that Jurkat cells, in which we detected FasL in detergent-resistant membranes, do not contain caveolae.57 It is unknown which of the FasL properties that change to rafts on recruitment is important for its increased killing capacity.
Fas ligand (FasL), a member of the TNF protein family, potently induces cell death by activating its matching receptor Fas. Fas-mediated killing plays a critical role in naturally and pathologically occurring cell death, including development and homeostasis of the immune system. In addition to its receptor-interacting and cell death–inducing extracellular domain, FasL has a well-conserved intracellular portion with a proline-rich SH3 domain–binding site probably involved in non-apoptotic functions. We report here that, as with the Fas receptor, a fraction of FasL is constitutively localized in rafts. These dynamic membrane microdomains, enriched in sphingolipids and cholesterol, are important for cell signaling and trafficking processes. We show that FasL is partially localized in rafts and that increased amounts of FasL are found in rafts after efficient FasL/Fas receptor interactions. Raft disorganization after cholesterol oxidase treatment and deletions within the intracellular FasL domain diminish raft partitioning and, most important, lead to decreased FasL killing. We conclude that FasL is recruited into lipid rafts for maximum Fas receptor contact and cell death–inducing potency. These findings raise the possibility that certain pathologic conditions may be treated by altering the cell death–inducing capability of FasL with drugs affecting its raft localization.
Identification of genes involved in resistance to interferon-α in cutaneous T-cell lymphoma
2002, American Journal of Pathology
Citation Excerpt :
CAV1, a caveolin underexpressed by IFN-resistant cells, is also a membrane raft protein. However MAL and CAV1 are found in distinct membrane microdomains/rafts,29 thus suggesting that they may have specialized functions. CAV1 is a tumor suppressor gene, localized on chromosome 7q31, for which a role as a negative regulator of the Ras-p42/44 MAP kinase cascade has been proposed (Figure 7).
Interferon-α therapy has been shown to be active in the treatment of mycosis fungoides although the individual response to this therapy is unpredictable and dependent on essentially unknown factors. In an effort to better understand the molecular mechanisms of interferon-α resistance we have developed an interferon-α resistant variant from a sensitive cutaneous T-cell lymphoma cell line. We have performed expression analysis to detect genes differentially expressed between both variants using a cDNA microarray including 6386 cancer-implicated genes. The experiments showed that resistance to interferon-α is consistently associated with changes in the expression of a set of 39 genes, involved in signal transduction, apoptosis, transcription regulation, and cell growth. Additional studies performed confirm that STAT1 and STAT3 expression and interferon-α induction and activation are not altered between both variants. The gene MAL, highly overexpressed by resistant cells, was also found to be expressed by tumoral cells in a series of cutaneous T-cell lymphoma patients treated with interferon-α and/or photochemotherapy. MAL expression was associated with longer time to complete remission. Time-course experiments of the sensitive and resistant cells showed a differential expression of a subset of genes involved in interferon-response (1 to 4 hours), cell growth and apoptosis (24 to 48 hours.), and signal transduction.
MAL Mediates Apical Transport of Secretory Proteins in Polarized Epithelial Madin-Darby Canine Kidney Cells
2001, Journal of Biological Chemistry
Citation Excerpt :
Cholesterol sequestration with CD inhibits secretion of Tg in MDCK cells, implicating lipid rafts in Tg transport (27). The same treatment did not affect the residence of MAL in rafts, probably because of its strong association with those specialized membranes as revealed by its resistance to solubilization even at a high concentration of detergent (36). In contrast, incorporation of newly synthesized Tg into rafts was greatly diminished by CD treatment, suggesting a weak association of the protein with the rafts.
The MAL proteolipid is an integral membrane protein identified as a component of the raft machinery for apical sorting of membrane proteins in Madin-Darby canine kidney (MDCK) cells. Previous studies have implicated lipid rafts in the transport of exogenous thyroglobulin (Tg), the predominant secretory protein of thyroid epithelial cells, to the apical surface in MDCK cells. We have examined the secretion of recombinant Tg and gp80/clusterin, a major endogenous secretory protein not detected in Triton X-100 insoluble rafts, for the investigation of the involvement of MAL in the constitutive apical secretory pathway of MDCK cells. We show that MAL depletion impairs apical secretion of Tg and causes its accumulation in the Golgi. Cholesterol sequestration, which blocks apical secretion of Tg, did not alter the levels of MAL in rafts but created a block proximal to Tg entrance into rafts. Apical secretion of gp80/clusterin was also inhibited by elimination of endogenous MAL. Our results suggest a role for MAL in the transport of both endogenously and exogenously expressed apical secretory proteins in MDCK cells.
Cell biology of caveolae and caveolin
2001, Advanced Drug Delivery Reviews
Originally described in the 1950s caveolae are morphologically identifiable as small omega-shaped plasma membrane invaginations present in most cell types. Caveolae are particularly abundant in adipocytes, fibroblasts, type 1 pneumocytes, endothelial and epithelial cells as well as in smooth and striated muscle cells. The first proposed function for caveolae was that of mediating the internalisation and transendothelial trafficking of solutes. Caveolae have been the object of intense research since the discovery of a biochemical marker protein, caveolin, in the early 1990s. Three genes encoding for caveolins have been characterised in mammals. Caveolins (18–24 kDa) are integral membrane proteins that constitute the major protein component of caveolar membrane in vivo. In addition to a structural role of caveolins in the formation of caveolae, caveolin protein interacts directly, and in a regulated manner, with a number of signalling molecules. We present here a general overview of the current knowledge on the structural role of caveolin in caveolae formation, and implication of caveolin in the control of cell signalling.

View all citing articles on Scopus

^☆: Abbreviations used: GPI, glycosylphosphatidylinositolMDCK, Madin-Darby canine kidney;

¹: To whom correspondence should be addressed. Fax: 34-1-397 8087. E-mail: [email protected].

View full text

Regular ArticleCaveolin and MAL, Two Protein Components of Internal Detergent-Insoluble Membranes, Are in Distinct Lipid Microenvironments in MDCK Cells☆

Abstract

Cell

Cell

Cell

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Anal. Biochem.

Curr. Opin. Cell Biol.

Science

J. Cell Biol.

Regular Article
Caveolin and MAL, Two Protein Components of Internal Detergent-Insoluble Membranes, Are in Distinct Lipid Microenvironments in MDCK Cells☆