(Received for publication, March 24, 1997, and in revised form, May 7, 1997)
From the Centro de Biología Molecular
"Severo Ochoa," Universidad Autónoma de Madrid, Consejo
Superior de Investigaciones Científicas, Cantoblanco, 28049 Madrid, Spain and ¶ The Whitehead Institute for Biomedical
Research, Cambridge, Massachusetts 02142-1479
The MAL proteolipid has been identified as a component of glycolipid-enriched membrane microdomains resistant to detergent solubilization in epithelial Madin-Darby canine cells, as well as in T lymphocytes and in myelin-forming cells. To study the function of the MAL proteolipid we have ectopically expressed a tagged form of MAL in both mammalian and insect cellular backgrounds. Immunofluorescence analysis in transiently transfected COS-7 cells showed the presence of MAL in large vesicular structures, and biochemical analysis identified MAL in the fraction of membranes resistant to Triton X-100 solubilization. Electron microscopic analysis showed that the expression of MAL in Sf21 cells morphologically resulted in the intracellular accumulation of large vesicles with a diameter from 200 to greater than 700 nm that were absent in uninfected or control infected cultures. Thus, ectopic expression of MAL in this heterologous expression system was sufficient to drive the formation of vesicles with a size similar to that of the vesicles detected in mammalian cells. These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression, as evidenced by co-infection experiments using a recombinant caveolin baculovirus. Taken together, these results suggest that the MAL proteolipid might play a role as a component of the machinery of vesiculation of glycolipid-enriched membranes.
Protein recruitment plays a role in a number of cellular processes including adhesion, signal transduction, and protein transport. The confinement of certain proteins into specialized membrane microdomains resistant to nonionic detergent (i.e. Nonidet P-40, Triton X-100) solubilization is emerging as one of the mechanisms used by the cell to recruit specific proteins (1-3). The high content in both glycolipids and cholesterol makes these microdomains insoluble in detergent (4). In epithelial Madin-Darby canine kidney (MDCK)1 cells, insoluble membranes have been found at the trans-Golgi network (TGN) and the plasma membrane (3, 5). It has been proposed that the membrane microdomains at the TGN are involved in the transport of glycosylphosphatidylinositol-anchored proteins, a limited number of transmembrane proteins, and glycolipids to the apical surface (1). According to this model, the self-association of glycolipids and cholesterol would form the biophysical basis for formation of the insoluble microdomains. However, to be operative as a route of transport, the glycolipid-enriched microdomains require protein sorting machinery that would minimally consist of a set of proteins to achieve the processes of vesicle formation, cargo recruiting, targeting, and fusion to the apical surface (1).
The MAL cDNA was initially identified during a search for genes
selectively expressed during T cell development (6). The MAL
gene is present in human chromosome 2 (7) and is organized into four
exons, each of which encodes a hydrophobic segment and an adjacent
short hydrophilic sequence (8). The MAL protein displays unusual
lipid-like properties that render MAL soluble in the organic solvents
commonly used to extract cell lipids (9). This feature allowed the
assignment of MAL to the proteolipid group, which includes other
proteins displaying similar lipid-like characteristics (10). More
recently, despite the restricted pattern of MAL gene transcription, MAL
expression has also been detected in epithelial MDCK cells (11) and
during the maturation of myelin-forming cells (12). Thus,
MAL gene expression is both tissue- and
differentiation-specific and appears to be modulated by elements distal
to its 5-proximal promoter region (13). In all of the cell types in
which is expressed, MAL has been identified as a component of
detergent-insoluble membrane microdomains (11, 12, 14). This fact, the
identification of MAL in TGN-related vesicles in epithelial cells (14),
and its predominance in apical transport vesicles in MDCK cells (11)
have led to the proposal that MAL is a component of the protein sorting
or vesiculation machinery of the glycolipid-mediated pathway of
transport (11, 14).
Here, we have approached the study of MAL function using overexpression of MAL in both COS-7 cells and Sf21 insect cells. Transient expression of MAL in COS-7 cells, as in other cell lines (11, 14), produces the accumulation of MAL in large vesicles. In this work, we have adopted the baculovirus expression system to study the possible vesiculation induced by MAL expression. Similar to the case in COS-7 cells, the expression of the MAL proteolipid in Sf21 insect cells induced the de novo formation of numerous intracellular vesicles ranging in size from 200 to more than 700 nm that almost completely filled the cytoplasm of the infected cells. These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression (15), as evidenced by co-infection experiments. The interaction of MAL with glycolipid-enriched membranes and its ability to generate extensive vesiculation suggest that MAL might be involved in vesiculation of the glycolipid-enriched microdomains.
The mouse hybridoma producing mAb 9E10 (IgG1) against the human c-Myc epitope EQKLISEED (16) was purchased from the American Type Culture Collection. Peroxidase-conjugated antibodies were from Pierce. Texas Red-conjugated antibodies were from Southern Biotech. Octyl glucoside and Triton X-100 were from Sigma. The baculovirus expression kit for recombinant baculovirus production was from CLONTECH.
Cell Culture ConditionsCOS-7 cells were grown at 37 °C in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (Life Technologies), penicillin (50 units/ml), and streptomycin (50 µg/ml) in an atmosphere of 5% CO2, 95% air. Insect Spodoptera frugiperda (Sf21) cells were provided by Drs. Takashi Okamoto and Ikuo Nishimoto (Massachusetts General Hospital/Harvard Medical School). Sf21 cells were grown at 27 °C in TC100 medium (Life Technologies) containing 10% fetal bovine serum (Life Technologies), penicillin (50 units/ml), and streptomycin (50 µg/ml).
DNA Constructions and TransfectionsThe human MAL cDNA
was tagged at its COOH terminus with sequences encoding the c-Myc
epitope and a polyhistidine (His7) tag by the polymerase
chain reaction using oligonucleotide primers corresponding to the 5-
and 3
-ends of the MAL coding sequence and full-length MAL cDNA (6)
as template. In addition, the primer corresponding to the 3
-end
contained sequences encoding the 9E10 c-Myc epitope and the
polyhistidine tag. For MAL expression in COS-7 cells, the polymerase
chain reaction product was subcloned into the pCR3 expression vector
(Invitrogen). Transfections were performed by electroporation using the
Electro Cell Manipulator 600 instrument (BTX, San Diego, CA)
For the generation of recombinant baculovirus, the MAL polymerase chain reaction product was subcloned into the pBacPAK9 transfer plasmid vector. A mixture of 2 µg of recombinant plasmid pBacPAK9-MAL and 1 µg of purified engineered baculoviral vector DNA BacPak6 linearized by Bsu36I digestion were co-transfected into Sf21 cells using Lipofectin-based standard protocols (Life Technologies, Inc.). Four days later, culture supernatants were removed and centrifuged at low speed. Clarified supernatants containing wild-type and recombinant baculoviruses were plaque-assayed on Sf21 cell monolayers. Occlusion-negative plaques were picked up and plated onto 2.5 × 106 cells. After 3 days of incubation, cells were collected and analyzed by immunoblotting using anti-c-Myc mAb 9E10. Viral plaques positive for MAL expression were selected and plated again. After three rounds of plaque purification, the plaque with the highest yield of MAL expression was used as recombinant baculovirus stock for subsequent experiments.
Characterization of MAL Expression in Sf21 Cells Using the Baculovirus SystemThe expression of MAL protein in the baculovirus system was evaluated by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Samples were separated by 15% acrylamide gels under reducing conditions and transferred to Immobilon-P membranes (Millipore). After blocking with 5% (w/v) nonfat dry milk, 0.05% (v/v) Tween-20 in phosphate-buffered saline (PBS), blots were incubated with mAb 9E10 culture supernatant at a ratio of 1:2 for 1 h, washed several times, and incubated with goat anti-mouse IgG antibodies coupled to horseradish peroxidase diluted at 1:5,000 in PBS/Tween 20. Blots were developed using an enhanced chemiluminescence Western blotting kit (ECL, Amersham).
Immunofluorescence MicroscopyCOS-7 cells grown on coverslips were washed with PBS, fixed in 4% (w/v) paraformaldehyde in PBS for 15 min, rinsed, and treated with 10 mM glycine in PBS for 10 min to quench the aldehyde groups. The cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min, rinsed, and incubated with 3% (w/v) bovine serum albumin for 20 min. Coverslips were then incubated with 9E10 mAb culture supernatant for 1 h, rinsed several times, and incubated for 1 h with goat anti-mouse IgG1 antibodies conjugated to Texas Red used at 1:1,000. After washing, the coverslips were mounted on slides. The cells were photographed with a Zeiss Axioskop photomicroscope using Kodak T-Max 400 film.
Transmission Electron MicroscopySamples were fixed with glutaraldehyde, postfixed with osmium tetraoxide, and stained with uranyl acetate and lead citrate, as detailed by Sargiacomo et al. (17) and Lisanti et al. (18).
Detergent Extraction ProceduresTriton X-100-insoluble complexes were separated by centrifugation to equlibrium in sucrose density gradients essentially as described by Brown and Rose (5) and Sargiacomo et al. (17). Cells grown to confluence in 100-mm dishes were rinsed with PBS and lysed with 1.8 ml of 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 at 4 °C. The lysate was scraped from the dishes with a rubber policeman, homogenized by passing the sample through a 22-gauge needle, brought to 40% sucrose (w/w) in a final volume of 4 ml, and finally placed at the bottom of an 8-ml 5-30% linear sucrose gradient. Gradients were centrifuged for 15-18 h at 39,000 rpm at 4 °C in an SW41 rotor (Beckman Instruments). Fractions of 1 ml were collected from the bottom of the tube, and aliquots were subjected to immunoblot analysis.
Velocity Gradient CentrifugationFor the estimation of the
oligomerization state of recombinant MAL, samples were loaded at the
top of a 5-40% linear sucrose gradient (4.3 ml) prepared in 25 mM Mes, pH 6.5, 150 mM NaCl buffer, and 60 mM octyl glucoside. After centrifugation at 50,000 rpm for
10 h in an SW60 rotor (Beckman Instruments), the gradient was
fractionated from the top. Aliquots from the different fractions were
subsequently subjected to immunoblot analysis using 9E10 mAb. Molecular
mass standards (Sigma) were as follows: carbonic anhydrase (29 kDa),
bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa),
-amylase (200 kDa), and apoferritin (443 kDa).
The
MAL proteolipid has been found as a component of detergent-insoluble
membranes in MDCK cells (11), oligodendrocytes (12), and T lymphocytes
(14). Transient expression of the MAL protein in mammalian cells
produces the accumulation of the MAL proteolipid in large vesicular
structures containing membranes resistant to detergent solubilization
(11, 14). We adopted a baculovirus system to express the MAL protein
tagged at its COOH terminus with a c-Myc epitope and a polyhistidine
tag in Sf21 insect cells. This expression system was used with the
double aim of (i) studying the possible vesiculation induced by MAL
expression and (ii) purifying MAL protein for in vitro
experiments. Our current model for MAL structure (8) as well as a
schematic of the tagged protein are shown in Fig. 1.
Although previous studies have established that tagging with either
c-Myc or polyhistidines does not generally interfere with the normal
properties of proteins, we first analyzed the incorporation of the
tagged MAL protein into detergent-insoluble membranes by centrifugation
to equilibrium and determined its presence in vesicular structures by
immunofluorescence analysis. Transiently transfected COS-7 cells were
extracted in 1% Triton X-100 at 4 °C, and the extracts were
subjected to centrifugation to equilibrium on sucrose density gradients
to separate lipid-rich resistant membranes from the Triton
X-100-soluble material (5). After gradient fractionation, aliquots from
the different fractions were subjected to immunoblot analysis using
9E10 mAb. Fig. 2 shows that MAL was confined to the low
density "floating" fractions (fractions 5-12), in agreement with
the reported presence of MAL in detergent-insoluble membranes (11, 12,
14). To analyze the doubly tagged MAL protein, COS-7 cells were
observed by immunofluorescence at 24 h post-transfection. Fig.
3 shows that MAL overexpression caused the accumulation
of the tagged protein along the exocytic pathway and in large vesicles at the cells' periphery.
To express the doubly tagged MAL protein in insect Sf21 cells, a
recombinant baculovirus was generated by standard techniques (19). A
band of the corresponding size of the tagged protein was specifically
detected in the infected samples when cell extracts from uninfected and
infected cells were analyzed by immunoblotting with 9E10 mAb (Fig.
4), demonstrating that the protein was efficiently expressed in Sf21 cells using the baculovirus system.
Expression of the MAL Proteolipid in Sf21 Cells Induces de Novo Formation of Large Intracellular Vesicles
Baculovirus-driven
expression of caveolin has been successfully used to generate
caveolae-like structures in the cytoplasm of Sf21 insect cells (15). We
have used this system to address whether expression of MAL can induce
vesicles as well. Electron microscopic analysis of uninfected cells
(Fig. 5A) or cells infected with a
baculovirus expressing control proteins (not shown) did not show any
remarkable difference in their cytoplasmic vesicle content. However,
MAL expression induced a massive de novo production of large
vesicles in the cytoplasm of the infected cultures (Fig. 5,
B-E). More than 100 vesicles were counted, and their sizes were determined. Fig. 5F shows that the range of vesicle
size was from 200 to more than 700 nm in diameter. Quantitative
analysis indicated that the major group (36% of the total number of
vesicles examined) includes vesicles with a diameter of ~300-400 nm,
whereas 80% of the vesicles have a diameter of ~200-600 nm.
Co-expression of MAL and Caveolin in Sf21 Cells Produces Different Populations of Vesicles
MAL and caveolin are two protein
candidates to be elements of the vesicular machinery for protein
sorting, operating in the glycolipid-mediated route of transport (11,
20). In addition, caveolin appears to function in the organization of
caveolar architecture (15, 21-23). Whereas caveolin expression in Sf21
cells induced the formation of vesicular structures with a diameter of
50-100 nm resembling intracellular caveolae (15), we have shown here that MAL expression induces much larger vesicles. To investigate whether there is any effect on the morphology of their respective vesicles under condition of simultaneous expression, we examined the
cytoplasm of cells co-infected with recombinant viruses expressing MAL,
caveolin-1, and caveolin-1
. Fig. 6 shows the
simultaneous presence in these cells of two different populations of
vesicles: large ones with a size corresponding to those induced by MAL
and small ones corresponding to the intracellular caveolae-like
vesicles induced by caveolin (15). This suggests that the vesiculation induced by MAL and caveolin are two separate processes.
Oligomerization of Recombinant MAL in Sf21 Cells
Caveolin is
known to form large homo-oligomers in both mammalian (24, 25) and
insect cells (15). To address whether MAL is also able to form
homo-oligomers, we assessed the oligomeric state of recombinant MAL in
insect cells by employing an established velocity gradient system (24).
Fig. 7 indicates that, in contrast to caveolin, MAL does
not form large oligomers, although dimers, trimers, and even tetramers
were detectable. Similar results were obtained in stably transfected
epithelial cells (not shown). This indicates that the generation of
vesicles induced by MAL expression does not require the formation of
large MAL homo-oligomers.
The identification of the components of the protein machinery involved in vesicle budding, transport, and fusion is a major focus in modern cell biology (26, 27). Especially challenging is the study of the mechanisms of vectorial transport in epithelial cells in which apical and basolateral plasma membrane proteins are sorted at the TGN by inclusion into separate vesicular carriers (28, 29). It has been proposed that one of the routes to the apical membrane in epithelial MDCK cells is mediated by glycolipid-enriched membrane clusters (1). One approach to dissect the protein machinery implicated in the glycolipid-mediated pathway of transport in MDCK cells has been the immunoisolation of the transport vesicles (30). Characterization of the proteins present in the detergent-resistant membrane fraction from apical vesicles has revealed the identity of four of these proteins: caveolin (VIP21) (20), VIP36 (31), annexin XIIIb (32), and MAL (VIP17) (11). It has been proposed that these four proteins might play a role as components of the vesicular transport machinery. Immunofluorescence analysis of transiently transfected epithelial (14) or COS-7 cells (this report) showed MAL in large ring-like structures at the times of massive overexpression of the protein, whereas a punctate pattern consisting of small vesicular structures is observed at earlier times.
The use of the recombinant baculovirus expression system allows a massive overexpression in every cell in the culture, making it possible to observe a great magnification of the effects produced by the protein; these effects are sometimes difficult to detect with lower levels of protein expression. A second advantage of this system is that it allows the analysis of the effects of ectopic protein expression in a nonmammalian protein background. To study the possible formation of vesicles induced by MAL expression, we developed an experimental system in which the human MAL proteolipid has been overproduced in Sf21 cells by using a baculovirus-based vector. Similar to the results of transient expression experiments in mammalian cells, MAL overexpression in the infected insect cells resulted in a large number of intracellular vesicles ranging in size from 200 to more than 700 nm in diameter. These vesicles were absent in infections with control baculoviruses as evidenced by electron microscopy analysis.
Caveolin is preferentially located in mammalian cells in specialized invaginations of the plasma membrane called caveolae (21) and consistently induces the formation of caveolae-like vesicles in insect cells (15). MAL, which is mainly located in TGN-derived vesicular structures (14), produces large vesicles in insect cells clearly different from those induced by caveolin as shown in co-infection experiments. It is plausible that both caveolin and MAL may belong to the vesicular transport machinery specific for the glycolipid-enriched microdomains but acting in the generation of different classes of vesicular carriers. Thus, is possible that more that one glycolipid-mediated route of transport can take place in the cell. For instance, caveolin-induced vesicles appear related to specific transport to caveolae, whereas MAL-induced vesicles might be involved in transport to the apical surface. The induction of two different types of vesicles by the simultaneous ectopic expression of MAL and caveolin in insect cells is in agreement with our recent results showing segregation of MAL and caveolin into distinct lipid microenvironments in MDCK cells (33).
Caveolin expression in insect cells induces intracellular caveolae-like vesicles but not surface caveolae (15). One interpretation of these findings is that these vesicles probably represent transient intermediates on the way toward fusion with the plasma membrane to form plasma membrane-attached caveolae. Thus, other factors absent in Sf21 cells may exist to connect these putative precursors to the plasma membrane. Similarly, MAL-induced vesicles in Sf21 cells were also intracellular, and we did not detect any fusion event between the vesicles and the plasma membrane. This suggests that the machinery for fusion of MAL-induced vesicles with the plasma membrane is not also operative in Sf21 cells. However, the heterogeneous size of the vesicles induced by MAL expression suggests that homotypic fusion events might occur, generating larger vesicles.
It has been shown that caveolin is found in mammalian cells as large homo-oligomers of up to ~400 kDa (24, 25). These oligomerization properties were preserved in insect cells expressing recombinant caveolin (15). On the contrary, the analysis of the oligomeric state showed that MAL does not form large oligomers but that it can be found as dimers, trimers, and even tetramers in both mammalian and insect cells. The interaction of MAL with itself and with selected endogenous lipid components might provide the basis for membrane vesiculation.
In summary, based on the specific presence of MAL in transport vesicles containing detergent-insoluble membranes (11, 14), MAL has been proposed to play a role as a component of the transport machinery of the glycolipid-mediated pathway. The results presented in this work showing the induction of extensive vesicle formation by MAL expression in insect cells are consistent with a role for MAL in vesiculation of glycolipid-enriched microdomains.
We thank members of Dr. Lisanti's and Dr. Alonso's laboratories for encouragement and helping discussions. We also thank Ya-Huei Tu (Whitehead Institute) for electron microscopy.