We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

REFERENCES

Akam
M.
(
1989
)
Hox and HOM: homologous gene clusters in insects and vertebrates.
Cell
57
,
347
349
Bally-Cuif
L.
,
Wassef
M.
(
1995
)
Determination events in the nervous system of the vertebrate embryo.
Curr. Opin. Genet. Dev
5
,
450
458
Benowitz
L.
,
Routtenberg
A.
(
1987
)
A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity.
Trends Neurosci
10
,
527
531
Berlose
J.
,
Convert
O.
,
Derossi
D.
,
Brunissen
A.
,
Chassaing
G.
(
1996
)
Conformational and associative behaviours of the third helix of antennapedia homeodomain in membrane-mimetic environments.
Eur. J. Biochem
242
,
372
386
Bouillot
C.
,
Prochiantz
A.
,
Rougon
G.
,
Allinquant
B.
(
1996
)
Axonal amyloid precursor protein expressed by neurons in vitro is present in a membrane fraction with caveolae-like properties.
J. Biol. Chem
271
,
7640
7644
Briata
P.
,
Di Blas
E.
,
Gulisano
M.
,
Mallamaci
A.
,
Iannone
R.
,
Boncinelli
E.
,
Corte
G.
(
1996
)
EMX1 homeoprotein is expressed in cell nuclei of the developing cerebal cortex and in the axons of the olfactory sensory neurons.
Mech. Dev
57
,
169
180
Chatelin
L.
,
Volovitch
M.
,
Joliot
A. H.
,
Perez
F.
,
Prochiantz
A.
(
1996
)
Transcription factor Hoxa-5 is taken up by cells in culture and conveyed to their nuclei.
Mech. Dev
55
,
111
117
Conrad
P. A.
,
Smart
E. J.
,
Ying
Y.-S.
,
Anderson
R. G. W.
,
Bloom
G. S.
(
1995
)
Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps.
J. Cell Biol
131
,
1421
1433
Derossi
D.
,
Calvet
S.
,
Trembleau
A.
,
Brunissen
A.
,
Chassaing
G.
,
Prochiantz
A.
(
1996
)
Cell internalization of the third helix of the antennapedia homeodomain is receptor-independent.
J. Biol. Chem
271
,
18188
18193
Derossi
D.
,
Joliot
A. H.
,
Chassaing
G.
,
Prochiantz
A.
(
1994
)
The third helix of Antennapedia homeodomain translocates through biological membranes.
J. Biol. Chem
269
,
10444
10450
Doe
C. Q.
,
Smouse
D.
,
Goodman
C. S.
(
1988
)
Control of neuronal fate by the Drosophila segmentation gene even-skipped.
Nature
333
,
376
378
Dotti
C. G.
,
Parton
R. G.
,
Simons
K.
(
1991
)
Polarized sorting of glypiated proteins in hippocampal neurons.
Nature
349
,
158
161
Dotti
C. G.
,
Simons
K.
(
1990
)
Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture.
Cell
62
,
63
72
Evan
G. I.
,
Lewis
G. K.
,
Ramsay
G.
,
Bishop
J. M.
(
1985
)
Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.
Mol. Cell. Biol
5
,
3610
3616
Faivre-Sarrailh
C.
,
Gennarini
G.
,
Goridis
C.
,
Rougon
G.
(
1992
)
F3/F11 cell surface molecule expression in the developing mouse cerebellum at synaptic sites and within granule cells.
J. Neurosci
12
,
257
267
Fra
A. M.
,
Williamson
E.
,
Simons
K.
,
Parton
R. G.
(
1995
)
De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.
Proc. Nat. Acad. Sci. USA
92
,
8655
8659
Gaunt
S. J.
,
Sharpe
P. T.
,
Duboule
D.
(
1988
)
Spatially restricted domains of homeo-gene transcripts in mouse embryos: relation to a segmented body plan.
Development
104
,
169
179
Gehring
W. J.
,
Qian
Y. Q.
,
Billeter
M.
,
Furukubo-Tokunaga
K.
,
Schier
A. F.
,
Resendez-Perez
D.
,
Affolter
M.
,
Otting
G.
,
Wuthrich
K.
(
1994
)
Homeodomain-DNA Recognition.
Cell
78
,
211
223
Gennarini
G.
,
Cibelli
C.
,
Rougon
G.
,
Mattei
M.-G.
,
Goridis
C.
(
1989
)
The mouse cell surface protein F3: a phosphatidylinositol-anchored member of the immunoglobulin superfamily related to chicken contactin.
J. Cell Biol
109
,
775
788
Gorodinsky
A.
,
Harris
D. A.
(
1995
)
Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin.
J. Cell Biol
129
,
619
627
Green
S.
,
Issemann
I.
,
Sheer
E.
(
1988
)
A versatile in vivo and in vitro eukaryotic expression vector for protein engineering.
Nucleic Acids Res
16
,
369
–.
Joliot
A.
,
Pernelle
C.
,
Deagostini-Bazin
H.
,
Prochiantz
A.
(
1991
)
Antennapedia homeobox peptide regulates neural morphogenesis.
Proc. Nat. Acad. Sci. USA
88
,
1864
1868
Keynes
R.
,
Krumlauf
R.
(
1994
)
Hox genes and regionalization of the nervous system.
Annu. Rev. Neurosci
17
,
109
132
Krämer
A.
,
Haars
R.
,
Kabisch
R.
,
Will
H.
,
Bautz
F.A.
,
Bautz
E.K.F.
(
1980
)
Monoclonal antibody directed against RNA polymerase II of DM.
Mol. Gen. Genet
180
,
193
199
Krumlauf
R.
(
1994
)
Hox genes in vertebrate development.
Cell
78
,
191
201
Kurzchalia
T. V.
,
Dupree
P.
,
Parton
R. G.
,
Kellner
R.
,
Virta
H.
,
Lehnert
M.
,
Simons
K.
(
1992
)
VIP21, a 21-kD membrane protein is an integral component of trans -Golgi-network-derived transport vesicles.
J. Cell Biol
118
,
1003
1014
Le Roux
I.
,
Duharcourt
S.
,
Volovitch
M.
,
Prochiantz
A.
,
Ronchi
E.
(
1995
)
Promoter-specific regulation of genes expression by an exogenously added homeodomain that promotes neurite growth.
FEBS Lett
368
,
311
314
Le Roux
I.
,
Joliot
A. H.
,
Bloch-Gallego
E.
,
Prochiantz
A.
,
Volovitch
M.
(
1993
)
Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.
Proc. Nat. Acad. Sci. USA
90
,
9120
9124
Lucas
W. J.
,
Bouche-Pillon
S.
,
Jackson
D. P.
,
Nguyen
L.
,
Baker
L.
,
Ding
B.
,
Hake
S.
(
1995
)
Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata.
Science
270
,
1980
1983
Miller
D. M.
,
Shen
M. M.
,
Shamu
C. E.
,
Burglin
T. R.
,
Ruvkin
G.
,
Dubois
M. L.
,
Ghee
M.
,
Wilson
L.
(
1992
)
C. elegans unc-4 gene encodes a homeoprotein that determines the pattern of synaptic input to specific motor neurons.
Nature
355
,
841
845
Mohr
E.
,
Richter
D.
(
1992
)
Diversity of mRNAs in the axonal compartment of peptidergic neuron in the rat.
Eur. J. Neurosci
4
,
870
876
Parton
R. G.
(
1996
)
Caveolae and caveolins.
Curr. Opin. Cell. Biol
8
,
542
548
Patel
N. H.
,
Martin-Blanco
E.
,
Coleman
K. G.
,
Poole
S. J.
,
Ellis
M. C.
,
Kornberg
T. B.
,
Goodman
C. S.
(
1989
)
Expression of engrailed proteins in arthropods, annelids, and chordates.
Cell
58
,
955
968
Prochiantz
A.
(
1996
)
Getting hydrophilic compounds into cells: lessons from homeopeptides.
Curr. Opin. Neurobiol
6
,
629
634
Prochiantz
A.
,
Theodore
L.
(
1995
)
Nuclear/growth factors.
BioEssays
17
,
39
45
Rijnboutt
S.
,
Jansen
G.
,
Posthuma
G.
,
Hynes
J. B.
,
Schornagel
J. H.
,
Strous
G. J.
(
1996
)
Endocytosis of GPI-linked membrane folate receptor-alpha.
J. Cell Biol
132
,
35
47
Rivera Pomar
R.
,
Niessing
D.
,
Schmidt
O. U.
,
Gehring
W. J.
,
Jackle
H.
(
1996
)
RNA binding and translational suppression by bicoid.
Nature
379
,
746
749
Rousselet
A.
,
Autillo-Touati
A.
,
Araud
D.
,
Prochiantz
A.
(
1990
)
In vitro regulation of neuronal morphogenesis and polarity by astrocyte-derived factors.
Dev. Biol
137
,
33
45
Rousselet
A.
,
Fetler
L.
,
Chamak
B.
,
Prochiantz
A.
(
1988
)
Rat mesencephalic neurons in culture exhibit different morphological traits in the presence of media conditioned on mesencephalic or striatal astroglia.
Dev. Biol
129
,
495
504
Rubenstein
J. R.
,
Puelles
L.
(
1994
)
Homeobox gene expression during development of the vertebrate brain.
Curr. Top. Dev. Biol
29
,
1
65
Skene
J. H. P.
,
Virag
I.
(
1989
)
Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43.
J. Cell Biol
108
,
613
624
Smart
E.
,
Ying
Y.
,
Donzell
W.
,
Anderson
R.
(
1996
)
A Role for Caveolin in the Transport of Cholesterol from Endoplasmic Reticulum to Plama Membrane.
J. Biol. Chem
271
,
29427
29435
Song
K. S.
,
Scherer
P. E.
,
Tang
Z.
,
Okamoto
T.
,
Li
S.
,
Chafel
M.
,
Chu
C.
,
Kohtz
D. S.
,
Lisanti
M. P.
(
1996
)
Expression of caveolin-3 in skeletal, cardiac and smooth muscle cells.
J. Biol. Chem
271
,
15160
15165
Tiedge
H.
,
Zhou
A.
,
Brosius
N. A. T.
(
1993
)
Transport of BC1 RNA in hypothalamo-neurohypophyseal axons.
J. Neurosci
13
,
4214
4219
Trembleau
A.
,
Morales
M.
,
Bloom
F. E.
(
1994
)
Aggregation of vasopressin mRNA in a subset of axonal swellings of the median eminence and posterior pituitary: light and electron microscopic evidence.
J. Neurosci
14
,
39
53
Vassar
R.
,
Chao
S. K.
,
Sitcheran
R.
,
Nunez
J. M.
,
Vosshall
L. B.
,
Axel
R.
(
1994
)
Topographic organization of sensory projections to the olfactory bulb.
Cell
79
,
981
991
White
J. G.
,
Southgate
E.
,
Thomson
J. N.
(
1992
)
Mutations in Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons.
Nature
355
,
838
841
This content is only available via PDF.