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Centro de Biología Molecular "Severo Ochoa" (F.M.-B., M.A.A.), Universidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049-Madrid, Spain; Departarment of Immunology and Oncology (L.K., J.P.A.), Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049-Madrid, Spain; and Department of Endocrinology (M.M.), Hospital de la Princesa, 28006-Madrid, Spain
Address all correspondence and requests for reprints to: Miguel A. Alonso, Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid, Spain. E-mail: maalonso{at}trasto.cbm.uam.es
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MAL complementary DNA (cDNA), initially identified during a search for genes differentially expressed during human T-cell differentiation (5), encodes a 17-kDa integral membrane protein containing several hydrophobic domains. The MAL protein displays lipid-like properties that render MAL soluble in the organic solvents commonly used to extract cell lipids. This unusual feature allowed the assignment of MAL to the proteolipid group of proteins (6). More recently, in addition to T cells, MAL expression has been detected in polarized epithelial MDCK cells (MAL/VIP17) (7) and in myelin-forming cells (MAL/MVP17) (8, 9), and MAL has been found in the GEM fraction in all of these cell types (7, 8, 10). The copurification of MAL with apically destined proteins in MDCK cells has led to the proposal of MAL as a component of the transport machinery for the GEM-mediated apical pathway (7). Moreover, the extensive de novo vesiculation induced by ectopic expression of MAL in a heterologous cell system suggests that MAL might be involved in GEM vesiculation (11).
Taking into account the proposed role of MAL as a component of the machinery for the apical pathway of transport mediated by GEMs, we have investigated the expression of MAL in the thyroid. Here, we describe that, despite the tissue-restricted pattern of MAL gene expression, MAL transcripts are present in the human thyroid gland and in the thyroid epithelial FRT cell line. Furthermore, immunohistochemical analysis of thyroid sections indicates that MAL is expressed in thyroid epithelial cells and that MAL distributes to the apical zone that faces the follicle lumen. Moreover, biochemical analyses demonstrate that MAL is a major integral membrane protein component of the GEM fraction of thyroid FRT cells, a model of polarized epithelial cell line which is devoid of thyroid specific functions. All these results are consistent with a role for MAL as a component of the GEM microdomains in thyroid epithelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture conditions and transfection
Rat FRT cells (13) (kindly provided by Dr. P. Santiesteban,
Instituto de Investigaciones Biomédicas, Madrid) were grown on
Petri dishes in F12 Coon’s medium (Sigma) supplemented with 10% FBS
(Gibco-BRL, Gaithersburg, MD), penicillin (50 U/ml), and streptomycin
(50 µg/ml), at 37 C in an atmosphere of 5% CO2/95% air.
Canine MDCK I cells were maintained under the same conditions in DMEM
supplemented with 10% FBS.
Transfection of FRT cells was carried out by electroporation using the Electro Cell Manipulator 600 equipment (BTX, San Diego, CA). Selection of stable transfectants was carried out by treatment with 0.5 mg/ml G-418 sulfate (Gibco-BRL) for at least 4 weeks after transfection. Drug-resistant cells were selected, screened by immuno-fluorescence analysis with 9E10 mAb, and the clones resulting positive for tagged MAL expression were maintained in drug-free medium. After several passages in this medium, more than 90% of the cells within the selected positive clones retained expression of tagged MAL.
Preparation of mAbs to human MAL
The peptide QDGFTYRHYH, corresponding to amino acids 114–123 of
the human MAL molecule, was synthesized on an automated
multiple-peptide synthesizer (AMS 422, Abimed, Langerfeld, Germany)
using the solid-phase procedure and standard Fmoc-chemistry (14). After
coupling to keyhole limpet hemocyanin, the peptide was used to immunize
BALB/c mice. Spleen cells from immunized mice were fused to mouse
myeloma cells, following standard protocols (15), and plated into
microtiter plates. The culture supernatants were screened by immunoblot
analysis using MAL-enriched membrane fractions prepared from epithelial
A498 cells stably expressing the MAL protein tagged with the 9E10 c-myc
epitope (10). The hybridoma clone 6D9 secreting antibodies to human MAL
was isolated after several rounds of screening and was used to produce
culture supernatants containing 6D9 mAb.
DNA constructions
Rat MAL cDNA was amplified with oligonucleotide primers
corresponding to the 5' and 3' untranslated regions of the rat MAL cDNA
sequence (8, 9) by PCR using cDNA synthesized by reverse transcription
of total RNA from FRT cells. The insertion of the 9E10 c-myc
epitope between the first and the second amino acid of MAL was carried
out by amplification of rat MAL cDNA by PCR with the oligonucleotide
primers N and C, which anneal to the 5' and 3' ends of the rat MAL
coding sequence, respectively. In addition, primer N contained
sequences encoding the 9E10 c-myc epitope placed between the first and
the second codon of the rat MAL coding sequence, and both N and C
primers contained one BglII site at their 5' end. After
amplification under standard conditions (16), the product was digested
with BglII and cloned into the unique BamHI site
of the pSR
expression vector (17). The construct expressing canine
MAL tagged with the 9E10 c-myc epitope was made, following identical
strategy, using RNA from MDCK cells. The sequence of the insert was
verified in all the constructs to eliminate the possibility of
amplification errors. The pSR constructs, expressing human MAL
tagged with the 9E10 c-myc epitope or mouse MAL tagged with the HA
epitope, have been previously described (10, 18). The construct
expressing recombinant human MAL in Escherichia coli cells
also has been described (6).
Northern blot analysis
The blot with poly(A)+ RNA (
2 µg per lane) from
different human endocrine tissues was purchased from Clontech (Palo
Alto, CA). Total RNA from different cell lines was extracted by cell
lysis with guanidinium thiocyanate, followed by a single-step
extraction with phenol (19). The polyadenylated RNA fraction was
purified by standard techniques (16). For Northern blot analysis of
different cell lines, approximately 5 µg poly(A)+ RNA
were denatured in 50% formamide and 2.2 M formaldehyde at
65 C, subjected to electrophoresis in a 1% agarose/formaldehyde gel,
and transferred to Nylon membranes. RNA samples were hybridized under
standard conditions, either to a human MAL cDNA fragment labeled by the
random-priming method or to an RNA probe generated by in
vitro transcription of the human MAL coding sequences (16). To
ensure that equal amounts of RNA were present in each lane, blots were
finally hybridized to a 0.6-kilobase (kb)
HinfI/BamHI DNA fragment from the 3' untranslated
region of human ß-actin messenger RNA (mRNA)(20). Final blot washing
conditions were 0.1 x SSC/0.1% SDS (1 x SSC = 0.15
M NaCl, 0.015 M sodium citrate, pH 7.0) at 65
C.
Detergent extraction procedures
GEMs were prepared essentially as described by Brown and Rose
(21) and Sargiacomo et al. (22). FRT cells grown to
confluency in 100-mm dishes were rinsed with PBS and lysed for 20 min
in 1 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl,
5 mM EDTA, 1% Triton X-100 at 4 C. The lysate was scraped
from the dishes with a rubber policeman, the dishes rinsed with 1 ml of
the same buffer at 4 C, and the lysate homogenized by passing the
sample through a 22-gauge needle. The lysate was finally brought to
40% sucrose (wt/wt) in a final vol of 4 ml and placed at the bottom of
an 8-ml 5–30% linear sucrose gradient. Gradients were centrifuged for
18 h at 39,000 rpm at 4 C in a Beckman SW41 rotor. Fractions of 1
ml were harvested from the bottom of the tube, and aliquots were
subjected to immunoblot analysis. Density was determined by measuring
the refractive index of the fractions. In some experiments,
centrifugation to equilibrium was carried out using discontinuous
sucrose density gradients consisting of a bottom 4-ml layer containing
the cell lysate in 40% sucrose, overlaid with 6 ml of 30% sucrose and
a 2-ml layer of 5% sucrose at the top. After centrifugation, the
opalescent band containing GEMs, which migrates in the 5–30% sucrose
interphase, was harvested from the top (fraction I). The 40% sucrose
layer, containing the cytosolic proteins and the solubilized proteins,
was harvested, as well (fraction S).
Immunoblot and immunoprecipitation analyses
For immunoblot analysis, samples were subjected to SDS-PAGE in
15% acrylamide gels under reducing conditions and transferred to
Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 5%
nonfat dry milk, 0.05% Tween-20 in PBS, blots were incubated with
either 9E10 or 6D9 mAb, used as culture supernatants, diluted 1:2.
After several washings, blots were incubated for 1 h with goat
antimouse IgG antibodies coupled to horseradish peroxidase diluted at
1:5,000, washed extensively, and developed using an enhanced
chemiluminescence Western blotting kit (ECL, Amersham, Buckinghamshire,
UK). For immunoprecipitation studies, extracts from metabolically
labeled cells were incubated for 4 h at 4 C with an irrelevant
control antibody bound to protein G-Sepharose and were centrifuged, and
the supernatant was immunoprecipitated by incubation for 4 h at 4
C with mAb 6D9 bound to protein G-Sepharose. Immunoprecipitates were
washed six times with 1 ml of 10 mM Tris-HCl, pH 8.0/0.15
M NaCl/1% Triton X-100, and were analyzed by SDS-PAGE
under reducing conditions. To detect 35S-labeling, dried
gels were finally exposed to Fujifilm imaging plates.
Immunohistochemistry
Surgical thyroid tissue was obtained from unaffected parts of
the thyroid gland of patients with localized carcinomas. Cryostat
sections were cut from snap-frozen thyroid tissue embedded in OCT
medium (Ames Co., Miles Laboratories, Elkhart, IN) stored at -80 C.
The tissue sections were stained by an indirect immunoperoxidase
method, as described previously (23). Briefly, 5-µm acetone-fixed
sections were sequentially incubated with 6D9 mAb culture supernatant
and peroxidase-conjugated rabbit antimouse Ig (Dako A/S, Glostrup,
Denmark). Each incubation was followed by three washes with Tris-HCl
buffered saline, pH 7.6. Then, sections were developed with
Graham-Karnovsky medium containing 0.5 mg/ml of 3,3'-diaminobenzidine
tetrahydrochloride (Sigma) and hydrogen peroxide. Sections were
counterstained with Carazzi’s hematoxylin, dehydrated, and mounted by
routine methods.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that the
thyroid gland expresses MAL mRNA species of the same size (1.1 kb) as
those present in human thymus, which served as a positive hybridization
control. MAL gene expression was undetectable in samples
from other human endocrine glands assayed (including pancreas, adrenal
cortex, adrenal medulla, and testis). To further investigate the
expression of the MAL gene, we used epithelial FRT cells, a
rat cell line of thyroid origin displaying a polarized phenotype (13).
Figure 1B shows that FRT cells express MAL mRNA species of 2.2 kb,
which is in agreement with the reported size of rat MAL mRNA (8, 9). As
positive hybridization controls, we included mRNA samples from Jurkat T
lymphocytes and epithelial MDCK cells, which express MAL transcripts of
1.1 and 1.0 kb, respectively, in accordance with the reported size of
the human and canine MAL mRNA species (24). No MAL transcripts were
found, either in human epithelial HeLa cells or human erythroleukemic
K562 cells or in mouse 3T3 fibroblasts, used as negative controls. This
pattern of expression was further confirmed by RT-PCR analysis. To
unambiguously identify MAL expression in FRT cells, MAL cDNA was cloned
from FRT cells and sequenced. The nucleotide sequence of the PCR
product (not shown) resulted in a sequence identical to that of rat MAL
cDNA previously deposited under the EMBL/GenBank accession number
X82557 (9).
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. A hybridoma clone (named 6D9),
producing antibodies to human MAL, was identified by immunoblot
analysis of membrane fractions enriched in MAL tagged with the 9E10
c-myc epitope (10). Figure 2B shows that the MAL decapeptide used for
the immunizations, at concentrations 
1 µg/ml, was able to
totally neutralize the recognition of the MAL protein by mAb 6D9,
whereas other control peptides did not present any effect (not shown).
The observed effect was specific for the 6D9 mAb, because the same
peptide did not influence the recognition of the tagged MAL protein by
anti-c-myc 9E10 mAb (Fig. 2B). To study the species specificity of the
6D9 mAb, tagged forms of human, rat, dog, and mouse MAL were
transiently expressed in COS-7 cells. Total cell lysates were prepared
24 h after transfection and assayed with either 6D9 or anti-tag
mAb. Figure 2C shows that 6D9 mAb recognizes human and rat MAL, but it
does not recognize the canine or murine protein or any endogenous
protein of COS-7 cells. The specific recognition of human and rat MAL,
but not of the murine protein, suggests that R120 is
crucial for the recognition of MAL by mAb 6D9 (see Fig. 2A). Thus,
substitution of R120 by K in dog MAL likely contributes to
the unreactivity of the canine protein with the 6D9 mAb.
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shows that, in agreement with the
presence of MAL transcripts in the thyroid, positive MAL staining was
found in thyroid epithelial cells. Moreover, MAL distribution was
confined to the apical zone delineating the follicle lumen, whereas no
staining was found at the basal or lateral membranes.
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), we adopted this cell line for biochemical analysis of the
MAL protein. The GEM fraction, which is resistant to solubilization by
nonionic detergent at low temperatures (21), was separated from the
bulk of cellular membranes, which are solubilized by the detergent, and
from cytosolic proteins, by using an established protocol involving
centrifugation to equilibrium on sucrose density gradients (21, 22).
FRT cells were extracted with 1% Triton X-100 at 4 C, and the extracts
were centrifuged to equilibrium. Twelve 1-ml fractions were obtained
after fractionation of the gradient from the bottom of the tube. The
top panel of Fig. 4A shows the
density expressed as a percentage of sucrose (wt/wt) in the different
fractions. Aliquots from each fraction were subsequently separated by
SDS-PAGE and stained with coomassie blue (Fig. 4, middle
panel). Fractions 1–4, which correspond to the 40% sucrose
layer, contain soluble proteins and represent more than 99% of the
total cellular proteins, as shown by gel staining. Fractions 5–12
contain GEMs that attain buoyancy in the 5–30% sucrose gradient, and
they represent less than 1% of the total cellular protein. When the
different fractions were analyzed by immunoblotting with anti-MAL 6D9
mAb, MAL was identified as exclusively present in the floating
membranes, indicating that endogenous MAL specifically resides in GEM
microdomains in FRT cells (Fig. 4A, bottom panel). Moreover,
to further study the ability of rat MAL to get targeted to GEMs, we
prepared FRT cell transfectants stably expressing the rat MAL protein
tagged at the N-terminus with the c-myc 9E10 epitope. Figure 4B shows
that exogenous rat MAL was correctly incorporated into GEM microdomains
in FRT cells.
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shows that, in contrast to the GEM fraction of MDCK cells, which
displays a large number of proteins (right panel), only a
weak protein band (of 17-kDa) was specifically detected in the same
fraction of FRT cells using exposure periods which caused
overexposition of the total and soluble fractions (right
panel). A second protein band of approximately 45-kDa was detected
in both the soluble and GEM fractions. This protein was identified as
actin by immunoblot analysis with specific antibodies (Fig. 5B), in
agreement with the reported association of actin with GEMs (25). The
17-kDa protein band specifically present in the GEM fraction of FRT
cells was identified as the MAL protein by both immunoblot and
immunoprecipitation analyses with anti-MAL mAb 6D9 (Fig. 5B).
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shows that the MAL protein was the
unique proteolipid detected in the GEM fraction of FRT cells, whereas
two major proteolipid bands were evident in the equivalent fraction of
MDCK cells. Minor proteolipid bands were detected in the MDCK cell
sample, but not in that of FRT cells, after longer exposures. One of
the major proteolipids in the MDCK cell sample had the same migration
as that of rat MAL and recombinant human MAL, and it likely corresponds
to canine MAL (7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A remarkable difference in the polarized transport between MDCK and FRT cells is that, whereas MDCK cells target both GPI-anchored proteins (27) and GlucCer (2) to the apical membrane, FRT cells sort them basolaterally (28, 29). The apical or basolateral surface distribution of endogenous transmembrane proteins has been found completely equivalent in MDCK and FRT cells (30), suggesting that GPI-anchored proteins use different pathways for apical transport than those used for the majority of transmembrane proteins. Most evidence supporting a role for GEM microdomains as a route of apical transport has been obtained in polarized epithelial MDCK cells (4). The GEM-mediated apical route has been previously compared in MDCK and FRT cells (29). FRT cells are able to assemble GEMs (22), but, contrary to MDCK cells, the GEM fraction from FRT cells seems to be depleted of GPI-anchored proteins (29). These findings were interpreted as indicating that the protein sorting machinery for GEM-mediated transport is incomplete in FRT cells and, as a consequence, this route is not operative in this cell line (29). Taking into account the proposed role of MAL as a component of the machinery for the GEM-mediated apical pathway of transport in MDCK cells, our results identifying MAL as a major integral membrane component of the GEM microdomains of FRT cells indicate that: 1) MAL is able to be specifically targeted to GEMs, whereas the majority of endogenous integral membrane proteins of FRT cells are not; and 2) MAL is not sufficient to recruit cargo proteins into GEMs.
Epithelial FRT cells display a polarized phenotype and tight junctions but lack thyroid-specific functional properties (13). In contrast, although epithelial FRTL-5 cells do exhibit thyroid-specific functions, they display limited polarity properties and lack tight junctions (31, 32), making them unappropriate for the study of polarized secretion. In addition, the FRTL-5 cell line does not express detectable levels of MAL mRNA or protein as assessed by Northern and Western blot analyses, respectively (not shown). Thus, none of these cell lines is a good model to address the possible role of the GEMs-containing MAL in vectorial transport of thyroid-specific molecules. The availability of the novel anti-MAL 6D9 mAb described in this work, together with the use of primary thyrocyte cultures, will be very helpful in the elucidation of the role of the GEMs containing MAL in thyroid cells.
Recombinant expression of either MAL or caveolin, another protein resident in the GEM fraction in some cell types, is able to induce intracellular vesicle formation (33). Interestingly, the vesicles induced by MAL are clearly different from the caveolae-like vesicles induced by caveolin, as evidenced by ectopic coexpression experiments in insect cells (11). This suggests that vesiculation induced by MAL and by caveolin are two separate processes. Thus, it is plausible that both caveolin and MAL may belong to the vesiculation machinery specific for GEM microdomains, but they act in the generation of different classes of vesicular carriers. This is in agreement with our recent results showing segregation of MAL and caveolin into distinct lipid microenvironments in MDCK cells (24). Because caveolin expression is absent in FRT cells (22, 29), our results demonstrate that MAL can access to GEMs independently of caveolin expression.
In this work, we report the expression of the MAL gene in the thyroid, the presence of MAL as a major protein component of the GEM fraction of epithelial thyroid FRT cells, and the distribution of MAL at the apical zone of normal thyroid epithelial cells in intact follicles. The availability, both of the novel anti-MAL mAb herein described and of molecular probes specific for the MAL gene (34, 35), opens up the possibility of addressing whether or not alterations in MAL expression take place in thyroid pathologies (1), such as carcinomas, in which epithelial polarity might be affected (36).
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Acknowledgments |
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Footnotes |
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Received August 4, 1997.
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promoter: an efficient and versatile
mammalian cDNA expression system composed of the simian virus 40 early
promoter and the R-U5 segment of human T-cell leukemia virus type 1
long terminal repeat. Mol Cell Biol 8:466–472This article has been cited by other articles:
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  A. Berchuck, E. S. Iversen, J. Luo, J. P. Clarke, H. Horne, D. A. Levine, J. Boyd, M. A. Alonso, A. A. Secord, M. Q. Bernardini, et al. Microarray Analysis of Early Stage Serous Ovarian Cancers Shows Profiles Predictive of Favorable Outcome Clin. Cancer Res., April 1, 2009; 15(7): 2448 - 2455. [Abstract] [Full Text] [PDF]
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 S. P. Ramnarayanan, C. A. Cheng, M. Bastaki, and P. L. Tuma Exogenous MAL Reroutes Selected Hepatic Apical Proteins into the Direct Pathway in WIF-B Cells Mol. Biol. Cell, July 1, 2007; 18(7): 2707 - 2715. [Abstract] [Full Text] [PDF]
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 J.-Y. Koo, I. Sohn, S. Kim, and J. W. Lee Structured polychotomous machine diagnosis of multiple cancer types using gene expression Bioinformatics, April 15, 2006; 22(8): 950 - 958. [Abstract] [Full Text] [PDF]
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 A. Llorente, M. C. de Marco, and M. A. Alonso Caveolin-1 and MAL are located on prostasomes secreted by the prostate cancer PC-3 cell line J. Cell Sci., October 15, 2004; 117(22): 5343 - 5351. [Abstract] [Full Text] [PDF]
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 M. Marazuela, A. Acevedo, M. A. Garcia-Lopez, M. Adrados, M. C. de Marco, and M. A. Alonso Expression of MAL2, an Integral Protein Component of the Machinery for Basolateral-to-Apical Transcytosis, in Human Epithelia J. Histochem. Cytochem., February 1, 2004; 52(2): 243 - 252. [Abstract] [Full Text] [PDF]
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M. Marazuela, A. Acevedo, M. Adrados, M. A. Garcia-Lopez, and M. A. Alonso Expression of MAL, an Integral Protein Component of the Machinery for Raft-mediated Apical Transport, in Human Epithelia J. Histochem. Cytochem., May 1, 2003; 51(5): 665 - 674. [Abstract] [Full Text] [PDF]
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 C. Copie-Bergman, M.-L. Boulland, C. Dehoulle, P. Moller, J.-P. Farcet, M. J. S. Dyer, C. Haioun, P.-H. Romeo, P. Gaulard, and K. Leroy Interleukin 4-induced gene 1 is activated in primary mediastinal large B-cell lymphoma Blood, April 1, 2003; 101(7): 2756 - 2761. [Abstract] [Full Text] [PDF]
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 F. Ulloa and F. X. Real Benzyl-N-acetyl-alpha -D-galactosaminide Induces a Storage Disease-like Phenotype by Perturbing the Endocytic Pathway J. Biol. Chem., March 28, 2003; 278(14): 12374 - 12383. [Abstract] [Full Text] [PDF]
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M. A. Alonso and J. Millan The role of lipid rafts in signalling and membrane trafficking in T lymphocytes J. Cell Sci., March 13, 2002; 114(22): 3957 - 3965. [Abstract] [Full Text] [PDF]
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R. Puertollano, J. A. Martinez-Menarguez, A. Batista, J. Ballesta, and M. A. Alonso An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells Mol. Biol. Cell, June 1, 2001; 12(6): 1869 - 1883. [Abstract] [Full Text] [PDF]
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F. Martín-Belmonte, R. Puertollano, J. Millán, and M. A. Alonso The MAL Proteolipid Is Necessary for the Overall Apical Delivery of Membrane Proteins in the Polarized Epithelial Madin-Darby Canine Kidney and Fischer Rat Thyroid Cell Lines Mol. Biol. Cell, June 1, 2000; 11(6): 2033 - 2045. [Abstract] [Full Text]
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C. Copie-Bergman, P. Gaulard, L. Maouche-Chretien, J. Briere, C. Haioun, M. A. Alonso, P.-H. Romeo, and K. Leroy The MAL Gene Is Expressed in Primary Mediastinal Large B-Cell Lymphoma Blood, November 15, 1999; 94(10): 3567 - 3575. [Abstract] [Full Text] [PDF]
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R. Puertollano and M. A. Alonso MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane Mol. Biol. Cell, October 1, 1999; 10(10): 3435 - 3447. [Abstract] [Full Text]
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 K. H. Cheong, D. Zacchetti, E. E. Schneeberger, and K. Simons VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells PNAS, May 25, 1999; 96(11): 6241 - 6248. [Abstract] [Full Text] [PDF]
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 R. Puertollano, F. Martin-Belmonte, J. Millan, M. del Carmen de Marco, J. P. Albar, L. Kremer, and M. A. Alonso The MAL Proteolipid Is Necessary for Normal Apical Transport and Accurate Sorting of the Influenza Virus Hemagglutinin in Madin-Darby Canine Kidney Cells J. Cell Biol., April 5, 1999; 145(1): 141 - 151. [Abstract] [Full Text] [PDF]
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R. Puertollano and M. A. Alonso A Short Peptide Motif at the Carboxyl Terminus Is Required for Incorporation of the Integral Membrane MAL Protein to Glycolipid-enriched Membranes J. Biol. Chem., May 22, 1998; 273(21): 12740 - 12745. [Abstract] [Full Text] [PDF]
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M. del Carmen de Marco, L. Kremer, J. P. Albar, J. A. Martinez-Menarguez, J. Ballesta, M. A. Garcia-Lopez, M. Marazuela, R. Puertollano, and M. A. Alonso BENE, a Novel Raft-associated Protein of the MAL Proteolipid Family, Interacts with Caveolin-1 in Human Endothelial-like ECV304 Cells J. Biol. Chem., June 15, 2001; 276(25): 23009 - 23017. [Abstract] [Full Text] [PDF]
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F. Martin-Belmonte, P. Arvan, and M. A. Alonso MAL Mediates Apical Transport of Secretory Proteins in Polarized Epithelial Madin-Darby Canine Kidney Cells J. Biol. Chem., December 21, 2001; 276(52): 49337 - 49342. [Abstract] [Full Text] [PDF]
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