The Third Helix of the Antennapedia Homeodornain Translocates through Biological

THE JDWRXAL OF BioLoCteAL CHEIMMTRY 0 1994 by The American Society for Biochemistry and Molecular Biolow, Inc.

Val. 269, No. 14, Issue of April 8, pp. 10444-10450, 1994 Printed in U S A .

The Third Helix of the Antennapedia Homeodornain Translocates through Biological ~embranes~

(Received for publication, June 14, 1993, and in revised form, January 3, 1994)

Daniele Derossit, Main H. Joliott, G6rard Chassaingl, and Main Prochiantztn From the $Centre National de la Recherche Scientifique Unite de Recherche Associee 1414, Ecole Normale SupQrieure, 46, rue d’Ulm, 75230 Paris Cedex 05, France and the $Centre National de la Recherche Scientifique Unite de Recherche Associee 493, Chimie Organique et B~olog~que, Uni~er$itQ Pierre et Marie Curie, 4, Place Jussieu, 75005 Paris, France

The 60-amino acid long homeodomain of Antennape- dia crosses biological membranes by an energy-independent mechanism, a phenomenon abolished by di- rected mutagenesis within the pol~eptide C-terminal region. This finding led us to study the internalization of several chemically synthesized peptides derived from the third helix of the homeodomain. We report here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N- terminal glutamate is still capable of translocating through the membrane. A longer peptide of 20 amino acids also translocates, whereas shorter peptides (16 amino acids) are not internalized by the cells. As is also the case for the entire homeodomain, the 20- and 16- amino acid long peptides are internalized at 4 “C, suggesting an energy-independent mechanism of tranloca- tion not involving ciassical endocytosis. The two translocated peptides can be recovered, intact, within the cells, strongly suggesting that they are not targeted to the lysosomal compartment. Finally, substitution of two tryptophans by two phenylalanines strongly dimin- ishes translocation, raising the possibility that the internalization of the third helix is not solely based on its general hydrophobicity.

Homeoproteins belong to a class of trans-activating factors first discovered in Drosophila and involved in multiple morphological processes (reviewed in Gehring (1987)). These proteins bind to DNA through a specific sequence of 60 amino acids, the homeodomain, which is structured in three a-helices with one p-turn between helices 2 and 3 (Qian et al., 1989). All verte- brates, including mammals, synthesize numerous homeoproteins that are phylogenetically related to proteins present in insects (reviewed in Holland, 1992). This phylogenetic kinship is striking at the level of the homeodomain and most particu- larly within the third a-helix. Homeoproteins are expressed in all embryonic derivatives, including the developing nervous system. Neural expression of h o m e o p ~ ~ i n s is very strong and appears to be quite prolonged, in some cases throughout adult- hood (Simeone et al,, 1992; Hunt et at., 1992; Price et at., 1992).

To understand the role of homeoproteins in post-mitotic neurons, we have recently developed a strategy based on the capability of the 60-amino acid homeodomain to bind to cognate

* This study was supported by Centre National de la Recherche Sci-

de Recherche sur le SIDA and Association Francaise de lutte contre les entifique, Ecole Normale SupBrieure, and grants from Agence Nationale

by the payment of page charges. This article must therefore be hereby Myopathies. The costs of publication of this article were defrayed in part

marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom correspondence should be addressed. Tel.: 33-1-44-32-37- 12; Fax: 33-1-44-32-39-88.

sequences present in target promoters and, thus, to antagonize the association of endogenous homeoproteins with these genomic targets (Joliot et al., 1991a). These experiments allowed us to observe, to our surprise, that the homeodomain of Antennapedia (that we called pAntp) is capable of translocating across the neuronal membranes and is conveyed to the nuclei (Joliot et al., 1991a, 1991b). This capture is energy- independent and provokes an increased morphological differ- entiation of the neurons (Joliot et al., 1991a, 1991b; Bloch- Gallego et al., 1993; Le Roux et al., 1993). We showed that pAntp mutants with no or little affinity for canonic homeodomain binding sites, yet still internalized, lack any biological activity. Therefore, the effect of pAntp on neuron morphology is entirely dependent on the specific DNA binding properties of the homeodomain (Le Roux et al., 1993).

In the course of these studies, we generated a mutant of pAntp ( p ~ t p 4 8 S ) in which, in addition to a Gln + Ser substitution in position 50 (within the third helix), two hydrophobic residues (Phe48 and Trp4’) were deleted (Le R o w et at., 1993). When we analyzed the translocating properties of the different peptides we observed that pAntp48S, in contrast to the wild- type homeodomain, is not internalized by nerve cells in culture (Le Roux et al., 1993). This finding suggests that the C-terminal region of the peptide and, in particular, its third helix might be important for the capability of the homeodomain to translocate through plasma membranes.

In order to examine this latter possibility, we have now chemically synthesized several peptides all related to the C terminus of p h t p . We demonstrate that a 16-amino acid long peptide that corresponds to the third helix deleted of its N- terminal glutamate is capable of translocating through biological membranes .

MATERIALS AND METHODS Peptide Synthesis-Peptide synthesis was carried out at a 0.1-mmol

scale (Applied Biosystems model 431A synthesizer) starting from an a Boc-Asn-PAM’ or CY Boe, c 2-Cl-Z Lys-PAM resine. All N-a-Boc amino acids, in 10-fold excess, were assembled using dicyclohexylcarbodiimide and 1-hydroxybenzotiazol as coupiing agents. After removal of the last N-a-Boc protecting group of 5-amino pentanoic acid, biotin was coupled as its N-hydroxy succinimido ester (4-fold excess, 15 h, dimethylform- amide). Peptides were cleaved from the resin by anhydrous fiuorhyobic acid and purifted by preparative reverse phase HPLC (Applied Biosys; tems), using a 10 x 250-nm Brownlee column (Aquapore Octyl, 300 A pore size) using various acetonitrile gradients in aqueous 0.1% triflu- oroacetic acid.

The purity of collected fractions was established by analytical isocratic separation (HPLC, Waters Associate) on Iichrosphere 10 RP-8 columns (Merck) and in 0.25 M triethylammonium phosphate, pH 3.0, (buffer A) and acetonitrile. Peptides were further analyzed by TLC

The abbreviations used are: Boc, t-butyloxycarbonyl; RPLC, high performance liquid chromato~aphy; TLC, thin layer ~hromato~aphy; PBS, phosphate-buffered saline.

10444

Danslocation of a Homeodomain Helix across Cell Membranes 10445

100

80

60

40

20

-1001 I80 190 200 210 220 230 240 2SO 260

Wavelength (nm) i" +3 101 5.9

I

600 800 1000 1200 1400 1600

mass spectrometry, and circular dichroism. Examples are illustrated for peptide 41-60.A, analytical isocratic separation (0.25 M triethylammonium FIG. 1. Analysis of peptide purity and a-helical structure. Peptide purity and structure were analyzed by analytical isocratic separation,

phosphate, pH 3, and 19.2% acetonitrile). B, molecular weight determination was performed by using an electrospray source combined with a quadripole mass spectrometer (R1010 Nermag). Under these conditions, multiple charged species are produced. Doubly and triply charged molecular species are detected, allowing exact mass determination by resolving the equations 1523.9 = (m + 2)/2 and 1015.9 = (m + 3)/3. C, circular dichroism spectra in water (dashed line) and in 30% HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (solid line).

using precoated silica gel plates (Merck) and butanol/pyridine/water/ acetic acid (5:5:4:1). Amino groups were detected with ninhydrin and biotin withp-dimethylaminocinamaldehyde. Peptide molecular weights were determined by Electrospray Ionization Mass Spectrometry in the laboratory of Dr. J . C. Tabet (Universite P. et M. Curie, Paris).

[Biotinylapa-Thr4l]pAntp(41-6O) = 4 1 4 0 purification (gradient:l2- 36% CH,CN, linear, 30 min) yield 22%, purity > 95% Electrospray: d z (M + nH)"' calcd. 3045.32 found 3045 TLC (R, = 0, 35) HPLC (is0 19.2% CH,CN in buffer A), 12.8 min [aIz5, = -32 "C (C, 0.5, 10% AcOH).

[Bi0tinylapa-Arg4~1pAntp(43-58) = 43-58 purification (gradient: 12- 22, 8% CH,CN, linear, 30 min) yield 17%, purity > 95% Electrospray: m/z (M + nH)"' calcd. 2571.86 found 2570 TLC (R, = 0.45) HPLC (is0

AcOH). 19.2% CH,CN in buffer A), 17.3 min [nIzSo = -10.7 "C (C, 0.5, 10%

[Bi~tinylapa-Arg"~, Phe@, Phe561pAntp(43-58) = 43-58FF purification (gradient:1230% CH,CN, linear, 30 min) yield 29%, purity > 95% Electrospray: m/z (M + nH)"' calcd. 2493.4 found 2492 TLC (R, = 0, 50)

AcOH). HPLC (is0 19.2% CH,CN in bufferA), 11 min [aIz5, = -16 "C (C, 0.5,10%

[Bi0tinylapa-Thr~~]pAntp(41-55) = 41-55 purification (gradient:l2- 33% CH,CN, linear, 30 min) yield 32%, purity > 95% Electrospray: m/z (M + n H Y calcd. 2359.52 found 2358 TLC (R, = 0, 45) HPLC (is0 19.2% CH,CN in buffer A), 15.4 min [aIz5, = -29.4 "C (C, 0.5, 10% AcOH).

[Biotinylapa-Ly~~]pAntp(46-60) = 46-60 purification (gradient12- 33% CH,CN, linear, 30 min) yield 16%, purity > 95% Electrospray: m/z (M + nH)"' calcd. 2417.58 found 2416 TLC (R, = 0, 42) HPLC (is0 19.2% CH,CN in buffer A), 6.8 min [(I]*~, = -30.1 (C, 0.5, 10% AcOH).

An illustration of the isocratic HPLC profile, mass analysis and circular dichroism spectra is given in Fig. 1 for the 20-amino acid long peptide. Circular dichroism was analyzed using a 1 cm pathlength quartz cell and a high sensitivity Jobin-Yvon dichrograph linked to a Minic Digital 11 miniprocessor. Peptide concentration was 26 p ~ . The (I helicity already visible in water (dashed lines) is strongly enhanced in 30% 1,1,1,3,3,3-Hexafluoro-2-propanol (solid line).

Peptide Analysis by Gel Filtrution-Gel filtration experiments were performed on a 30-cm long TosoHaas G 2000 SW, column using, as eluent, 0.1 M Tris, pH 6.8, with or without 0.1% SDS. The flow rate was 0.8 mumin. Molecular weight markers were purchased from Sigma.

Peptide Analysis by Polyacrylamide Gel Electrophoresis-Peptides a t the indicated concentrations or extracted from the cells (see below) were boiled in Laemmli buffer and run on a 12-22% polyacrylamide SDS gradient gel. Separated peptides were either stained directly in Coom- assie Blue or electrotransferred on Immobilon (Millipore) in 25 m~ Tris, 192 m~ glycine, pH 8.8. After a 2-h blocking in 20 m~ Tris-HC1, pH 7.5, 150 m~ NaCI, 150 m~ Tween 20, and 4% bovine serum albumin, the blots were incubated for 1 h at room temperature with streptavidin-

10446 Danslocation of a Homeodomain Helix across Cell Membranes peroxydase (Amersham Corp.) diluted 1:lOOO in the blocking solution with 0.5% bovine serum albumin, washed several times with the same solution, and revealed with luminol, according to the instructions of the manufacturer ( h e r s h a m ) .

In some cases, the peptides were loaded on a 12-22% gradient polyacrylamide acidic gel devoid of SDS according to Reisfeld et al. (1962). This technique allowed to separation of the peptides in the absence of SDS.

Cell Cultures-Cell culture conditions have been described in earlier reports (Lafont et al., 1992). Briefly, small fragments of tissues taken from different regions of the embryonic rat brain between E13 and E15 were incubated in trypsine (0.25%, Life Technologies, Inc.) for 5 min a t 37 "C, washed twice in phosphate buffer complemented with 33 mM glucose (PBS), and incubated for 10 min a t 37 "C with 15 pg/ml DNase I (Sigma) in Dulbecco's modified Eagle's medium/F12 supplemented with 33 m~ glucose, 2 mM glutamine, 10 mM Hepes pH 7.4, 3 mM NaHCO,, 5 unitdml penicillin and 5 pg/ml streptomycin. The cells were dissociated mechanically, washed 3 times in serum-free medium and plated a t a concentration of 25,000 celldcm2 on glass coverslips (16 mm diameter) coated with 15 pg/ml D,r,-polyornithine (Sigma). All cultures were in chemically defined medium consisting of serum-free medium supplemented with 0.1% ovalbumin, 100 pg/ml transferin, 20 nM pro- gesterone, 20 p~ putrescine, and 30 nM selenium.

Peptide Internalization and Visualization-When added to cells in culture, the peptides were diluted in chemically defined medium, and the volume added was equal to the volume of medium present in the culture dish. After 2-4 h, the cells were washed 3 times with chemically defined medium, fixed for 5 min a t -20 "C in ethanoVCH,COOH (95/5), washed 3 times in PBS, and incubated for 30 min with fluoresceinated streptavidin (Amersham) diluted 1000-fold in PBS plus 10% newborn calf serum (Life Technologies, Inc.). At the end of the incubation, the cells were washed 3 times in PBS, dried and, mounted in Mowiol before examination.

In some experiments, freshly dissociated cells were resuspended in 0.6 ml of PBS or chemically defined medium containing appropriate peptide concentrations plus the following mixture of protease inhibitors: 0.5 mM pefablock, 1 pg/ml a-2 macroglobulin, and 10 pg/ml leu- peptin. After 2 4 h of incubation with regular gentle shaking, the cells were centrifuged and washed 3 times with 1 ml of PBS plus protease inhibitors, and the final pellet was resuspended in Laemmli buffer, boiled, and frozen or directly loaded on a 12 to 22% polyacrylamide SDS gradient gel.

Confocal Microscopy-Data were obtained with a confocal scanning laser microscope Sarastro 2000 (Molecular Dynamics). Excitation was obtained with an Argon ion laser set at 488 nm for fluorescein isothio- cyanate excitation, and the emitted light was filtered with an appropriate long pass filter (510 nm). Sections presented were taken approximatively at the mid-height level of the cells. Photomultiplyer gain and laser power were identical within each experiment.

RESULTS

Fig. 2 shows the sequences of the five peptides used in this study and their relation with the third helix of pAntp. In addition to the amino acids present in the natural molecules, we have added, on the N-terminal side, an amino pentanoic acid spacer and a biotin residue that was used to follow the fate of the peptides. Among these peptides, the third one (43-58FF) corresponds to the replacement of two tryptophan residues present in positions 48 and 56 of the wild-type 43-58 peptide by two phenylalanines.

When the electrophoretic behaviors of the different peptides were analyzed on 12-22% SDS-polyacrylamide gradient gels, we consistently observed that in the presence of SDS, all peptides tended to form aggregates. This tendency is illustrated in Fig. 3A for the four peptides corresponding to a fragment encompassing the third helix (lane 1 ) or to different parts of the third helix (lanes 2 4 ) . This ability to form aggregates seems to be due to the presence of SDS, since no aggregates were visible when the peptides were run on an acidic gel devoid of SDS as illustrated in Fig. 38 for peptides 41-60 and 46-60. Accord- ingly, HPLC gel filtration elution profile of 41-60 peptide in absence of SDS (Fig. 3C) is consistent with the molecular weight of a monomer, whereas, in the presence of 0.1% SDS (Fig. 3D), the peptide coeluted with aprotinin, thus with a

piotinykpa-Lflphtp (46so)34sao

B i d - A p a l y s - l l ~ T T r p P h e G l n - A s n - A r g - A r g " l - L ~ - T ~ L ~ L ~ ~ l u - ~ n 46 60

in this study. Apa, amino pentanoic acid; Biot, Biotin. The region FIG. 2. Primary sequences of the different polypeptides used

corresponding to thehtennapedia homeodomain third helix is shaded.

molecular weight corresponding to that of a dimer. I t is note- worthy that dimeric forms are also predominant in SDS-polyacrylamide gel electrophoresis analysis. Dimerization of small peptides in SDS-polyacrylamide gel electrophoresis, though surprising, has already been reported by Lemmon and co-work- ers (1992a, 1992b) who raised the possibility that peptide behavior in a detergent environment and within a lipid bilayer might share similarities (Lemmon et al., 1992a, 1992b).

We first compared the behavior of the four helix 3-derived peptides added to neuronal cultures prepared from the nervous system of E15 rat embryos and kept for 2 days before peptide addition. Peptides added to an external concentration of 25 p~ were left in contact with the cells for 3.5 h a t 37 "C. The cells were then washed 3 times with culture medium, fixed, and incubated with fluorescent streptavidin. Fig. 4 gathers four confocal microscopy sections obtained at low magnification. It demonstrates that peptides 41-60 (Fig. 4A) and 43-58 (Fig. 4B ) are internalized and suggests a strong biotin accumulation at the level of the nuclei. In contrast, the two shorter peptides, 46-60 (Fig. 4C) and 41-55 (Fig. a), do not seem to be internalized.

To confirm the internalization of peptides 41-60 and 43-58, 1.5 x lo6 nerve cells freshly dissociated from the striata of E15 rat embryos were incubated for 2.5 h at 37 "C in 0.6 ml of culture medium containing the different peptides a t a concentration of 70 p ~ . At the end of this incubation period, the cells were centrifuged, and 30 pl of the incubation medium were kept for gel analysis. The cells were washed 3 times with 1 ml of culture medium and finally resuspended in 60 pl of gel loading buffer of which 30 pl were analyzed by gel electrophoresis and electrotransfer.

Fig. 5A shows that cells incubated with peptides 41-60 (lane 1) and 43-58 (lane 2) have internalized the two peptides and that these peptides are recovered undegraded. In contrast, the two shorter peptides (lanes 4 and 5) were not present in the cell extracts. The analysis of the culture medium (Fig. 5B) demonstrates that all peptides were present in similar amounts in the supernatant of the cells after the first centrifugation. Interest- ingly, peptides recovered after internalization formed multim- eric complexes. In addition to SDS-induced dimerization (see


B 1 2 3 4 1 2

minutes

D

.. minutes

"

FIG. 3. Gel analysis of the peptides and interaction with SDS. A, approximatively 3 pg of each peptide were boiled in Laemmli loading buffer, run on an SDS-containing 12-22" polyacrylamide gel gradient, transferred on Immobilon membranes, and incubated with streptavidin-peroxydase. Peroxydase activity was revealed by chemoluminis- cence. Lane 1, peptide 41-60; lune 2, peptide 43-58; lane 3, peptide 41-55; lune 4, peptide 46-60. Note that all peptides have a tendency to form aggregates. B, acidic electrophoresis (no SDS) of 16 pg of peptides

above), it is possible that aggregation is enhanced by the presence of lipids that would associate with the peptide during translocation. A nonexclusive possibility is that only aggre- gated peptides are capable of translocation. These possibilities are presently under investigation.

The internalization of the peptides is further demonstrated by their protection against the action of trypsine as illustrated in Fig. 6 for peptide 43-58. In this experiment, lofi cells were prepared and incubated as in Fig. 5, with the exception that the peptide in the culture medium was treated for 15 min a t 37 "C with 0.25% trypsine either before ( lane 1) or after incubation (lane 2 ) with the cells. Obviously, internalization protects the peptide against the action of the protease. Identical results were obtained with peptide 41-60 (not shown).

To verify whether peptides 41-60 and 43-58 behaved simi- larly to the entire homeodomain and were internalized by an energy-independent mechanism, we incubated the peptides with the cells as in Fig. 3, but, in some cases, the cells had first been cooled to 4 "C, and this temperature was maintained during the entire period of incubation. As illustrated in Fig. 7, the internalization of peptide 43-58 (and also of peptide 41-60) (not shown) was only moderately inhibited a t low temperature (compare Fig. 7, A and C ) . In contrast, lowering the temperature completely abolished the internalization of the small amounts of peptides 41-55 and 46-60 (not shown) endocytosed a t 37 "C (compare Fig. 7, B and D).

In all experiments described above, the peptides were used a t rather high concentrations (between 25 and 100 PM). We thus analyzed the influence of the external peptide concentration on the internalization of peptide 43-58 by freshly dissociated nerve cells. Cells were incubated for 2.5 h with concentrations ranging from 1.25 to 20 PM. Fig. 8 illustrates that increasing the external concentration does increase the amount of internalized peptide but that translocation occurs a t all concentrations tested.

Finally, we compared the behavior of peptide 43-58 and of its mutant version 43-58FF. Fig. 9 illustrates that when added a t an identical concentration of 25 PM, the two peptides presented very different internalization capacities, the wild-type version being internalized with a much higher efficiency (Fig. 9A) than the double mutant (Fig. 9B ).

This difference was confirmed by gel analysis. Fig. 10 allows comparison of the amounts of 43-58 ( lane 1 ) and 43-58FF (lane 3) recovered within the cells. Purified peptides were run on the same gel (lanes 4 and 5). This latter experiment also indicates that compared with 43-58,43-58FF has only a weak tendency to form aggregates, probably because it interacts less strongly with SDS micelles.

DISCUSSION

In this study, we demonstrate that a peptide of 16 amino acids included within the third helix of the Antennapedia homeodomain is internalized by neural cells in culture by an energy-independent mechanism. This peptide thus reproduces the behavior of the entire homeodomain reported in previous studies (Joliot et al . , 1991a, 1991b; Bloch-Gallego et al . , 1993;

41-60 (lane 1 ) and 46-60 (lane 2) ; note that both peptides migrate as a single band. C and D, gel filtration experiments, peptide 41-60 (molecular weight, 3,045) and molecular weight markers were eluted in 0.1 M Tris, pH 6.8, without ( C ) or with (D) 0.1% SDS. In absence of SDS, peptide 41-60 retention time corresponds to that expected for a mono- mere, whereas, in the presence of 0.1% SDS, the peptide is coeluted with Aprotinin (molecular weight, 6,500). thus with a mass compatible with that of a dimer. Vertical arrows indicate retention times of Blue dextran (molecular weight, 2,000,000). Albumin (molecular weight, 66,000), and cytochrome c (molecular weight, 12,400). The near elution of Blue dextran (2,000,000) and Albumin (66,000) in presence of SDS corresponds to the physical properties of the column provided by the manufacturer.

10448 Danslocation of a Homeodomain Helix across Cell Membranes

FIG. 4. Peptide visualization by confocal microscopy. Cells dissociated from E15 rat mesencephalon were cultured for 2 days a t a density of 25,000 cells/cm2 before addition of the different peptides a t a concentration of 25 p ~ . After 3.5 h, the cells were rinsed and fixed, and the presence of biotin was revealed with fluorescent streptavidin. The sections presented here demonstrate that biotin linked to peptides 41-60 (A) and 43-58 (B) is internalized. In contrast, biotin linked to peptides 46-60 (C) and 41-55 (Dl is not significantly internalized. Bar, 20 pm.

C

1 2 3 4 5 1 2 3 4 5 1 2

A B FIG. 5. Gel electrophoresis of culture medium and cell ex-

tracts. E15 striatal cells (1.2 x lo6 cells in 0.6 ml) were incubated with the different peptides a t a concentration of 70 p~ for 2.5 h a t 37 “C. After several washes, the extracellular and intracellular presence of the peptides was analyzed by gel electrophoresis and electrotransfer. The amounts loaded correspond to 50% of the total cellular extracts (A) and to 5% of the culture medium (B). Lanes I , peptide 41-60; lanes 2, peptide 43-58; lanes 3, empty; lanes 4, peptide 41-55; lanes 5; peptide 46-60. Note that although equal amounts of all peptides were present in the culture medium, only the two longer peptides were significantly internalized. Note also that all peptides have a strong tendency to form multimers.

Le Roux et al., 1993). In comparison, shorter versions of the third helix as well as a modified version were either poorly internalized or not internalized at all by the cells. This finding confirms the results of Le Roux et al. (1993) and strongly suggests that the driving force for homeodomain internalization resides in its third helix. It thus opens the way to a structure- function study of the phenomenon.

If we compare peptides 41-60 and 43-58 with the entire homeodomain, it appears that they have a similar behavior, since they translocate through the membranes at 37 and 4 “C and can diffuse or be conveyed toward the cell nuclei. However, given the extracellular concentrations of peptides used in this study and in studies involving the entire homeodomain, it

degradation. E16 cortical cells ( lofi in 0.6 ml) were incubated for 2 h a t FIG. 6. Internalized peptides are protected against proteolytic

37 ’C with peptide 43-58 a t a concentration of 100 p~ and analyzed by gel eletrophoresis and electrotransfer. In lane 1, the medium plus peptide were incubated with trypsine for 15 min before trypsine inhibition and addition to the cells. In lane 2, trypsine treatment was done at the end of the incubation period. In both cases, the cells were rinsed thor- oughly and resuspended in 100 pl of Laemmli loading buffer. After boiling, one-third of the cell extract was run. Note that the internalized peptide is protected against proteolytic degradation.

seems that the homeodomain (pAntp) crosses biological membranes with a higher efficiency. In fact, in most studies involving pAntp, we found a strong accumulation of the homeodomain in the cell nuclei even when the 60-amino acid long polypeptide was added to the cells a t an extracellular concentration of 5 x lo-’ M (Joliot et al., 1991b). This contrasts with the fact that the small penetrating peptides must be added a t concentrations higher than M. We indeed exclude an unspecific diffusion of the molecules due to membrane damage or passive leakage since shorter or slightly modified versions of the peptides are not internalized.

Thus, compared with that ofpAntp, the lower internalization of the small peptides could be explained by a “helper” effect of the two other helices during translocation. Another possibility is that the entire homeodomain binds more strongly to target


FIG. 7. Influence of temperature on peptide internalization. Peptides 43-58 (A and C) and 41-55 (B and D) were added a t a concentration of 25 p~ to E15 mesencephalic cells grown for 2 days a t a density of 25,000 cells/cm2. The incubation was done for 3.5 h a t 37 (A and B) or 4 "C ( C and D). Note that in this confocal microscopy section, the internalization of 43-58 is slightly reduced but not abolished a t 4 "C, whereas the small internalization of 41-55 that occurs at 37 "C is completely blocked a t the lower temperature. Bar, 20 pm.

1 2 3 4 5 6 7

FIG. 8. Dose-response internalization of peptide 43-58. Increas- ing concentrations of peptide were incubated in suspension for 2.5 h with lofi spinal cord neurons in a final volume of 0.6 ml. At the end of the incubation period, the cells were rinsed three times and an aliquot of 12 out of 50 pl of total extract was run on gel and electrotransferred. Lane 1, 1.5 pg of purified peptide; lunes 2 to 6 correspond to the incubation of the cells with 20, 10, 5, 2.5, and 1.25 p~ of peptide, respec- tively. Lane 7, no peptide, indicating that the parasite bands present on the blot are intrinsic to the cells and not due to the peptide.

genomic sequences and that, consequently, an extracellular- intracellular gradient favorable to an inward peptide current is maintained.

Indeed, to be valid, this explanation requires that the transport cannot be saturated. This was in fact verified by diluting a small concentration of radioactive homeodomain with increasing concentrations of the nonlabeled polypeptide, a protocol that leads to cell death before any plateau of internalized pAntp can be reached.' Finally, we cannot completely exclude that the differences observed between the complete homeodomain and the small peptides are artifactual and due to loss of the shorter peptides during the fixation procedure or the electrotransfer.

* D. Derossi, A. H. Joliot, G. Chassaing, and A. Prochiantz, unpub- lished experiments.

The absence of saturable transport and the internalization at 4 "C suggests that the polypeptides do not bind a transporter. In fact, their mode of translocation remains unclear, and several hypotheses can be considered. The observation that the double phenylalanine peptide of the same length, charge, and amphiphilicity as the unmodified third helix translocates with much lower efficiency and shows limited aggregation in SDS- gel electrophoresis experiments suggests that aggregation within the lipid bilayer and the formations of micelles or pores might be necessary in transport. More experiments are necessary to study these and other possibilities. However, aggregation alone cannot account for internalization in view of the absence of translocation of the 15-amino acid long peptides.

If we compare the internalization of the homeopeptides with that of other polypeptides also targeted to the cytoplasm, in particular the diphteria toxin A subunit, a main difference is that diphteria toxin A crosses the membranes at low pH range, either at the level of the plasma membrane if the pH value is artificially lowered or at the level of the endosomes where the pH value is physiologically low (Moya et al., 1985; Olsnes et ul., 1988; Moskaug et al., 1991; Jiang et al., 1991; Beaumelle et al., 1992; Papini et al., 1993), whereas pAntp or its third helix tranlocates at higher pH range, thus not requiring endocytosis.

This difference between the two classes of molecules could be due to the existence of completely different mechanisms of transmembrane translocation. Alternatively, it could be pro- posed that homeodomain translocation also requires a pH transition but that this transition is not as strong as that required for the translocation of diphteria toxin A. In fact, the necessity for a low local pH at the cell surface might explain why, although internalized by all cell types, the entire homeodomain is preferentially captured by cells expressing high levels of poly- sialic acid a t their surfaces (Joliot et al., 1991b). In the case of the small peptides, we have verified that they were internalized by all cell types, but we have not yet compared the respec- tive efficiency of the different cells nor the effect of pH value on the internalization.

10450 Banslocation of a Homeodomain Helix across Cell Membranes

FIG. 9. Peptide 43-68FF is poorly translocated (confocal microscopy). Peptides 43-58 and 43-58FF were added a t a concentration of 25 p to E13 spinal cord cells (25,000 celldcm*) cultured for 2 days. After 2.5 h, the cells were washed, fixed, and analyzed by confocal microscopy. Peptide 43-58 (A) is internalized at a much higher rate than the double phenylalanine mutant (B). Bar, 20 pm.

1 2 3 4

FIG. 10. Peptide 4368FF is poorly translocated (gel analysis). E14 spinal cord cells (lofi in 0.6 ml) were incubated for 2 h at 37 "C with 43-58 or 43-58FF a t a concentration of 50 w. Following incubation, the cells were washed 3 times and resuspended in 50 pl of Laemmli buffer, and 25 pl of the extracts were run on a gel and electrotransferred. Lane

43-58 (1.5 pg); lane 5, purified 43-58FF (1.5 pg). The star indicates the 1, 43-58; lane 2, no peptide added; lane 3, 43-58FF; lane 4, purified

level of migration of a 14.4-kDa molecular weight marker that reacted slightly with streptavidin-peroxydase.

Other polypeptides that cross biological membranes are those destined, after synthesis, to specific intracellular com- partments such as the endoplasmic reticulum or the mitochon- dria (Rapoport, 1992; Glick et al., 1991). Passage through these intracellular membranes is energy-dependent and requires the presence of specific proteins that serve as receptors and/or channels. However, even in this rather well studied system, the actual mechanism of importation is not yet completely under- stood. Indeed, a basic fragment of Dynorphin B (Dynorphin B 1-13), which does not antagonize the import of the presequence of cytochrome c oxydase subunit IV (pCyt OX IV), is able to translocate through the outer mitochondrial membrane a t 4 "C (25). This latter result suggests that translocation through the outer membrane could be achieved through several distinct mechanisms. Furthermore, the recent report by Maduke and b i s e (19931, suggesting that pCyt OX IV is imported into pro- tein-free phospholipid vesicles, challenges the current view on the mandatory role of proteinacious binding sites and channels, a t least for the crossing of the outer mitochondrial membrane.

The physiological significance of the phenomenon is un- known. The most straightforward possibility is that, although real, homeodomain internalization is an interesting and poten- tially useful artifact. A second possibility is that it reflects the capability of homeoprotein isoforms or fragments to be released from cells and passaged into other cells through a paracrine mechanism.

We cannot presently answer this question, but two points can be raised. First, fusion proteins up to 109 amino acids in length and encompassing the entire homeodomain are internalized

and addressed to the cytoplasm and nucleus, suggesting that rather large polypeptides do have the possibility to translocate (Perez et al., 1992). Second, the present study, as well a previous study in which we analyzed the translocation property of different mutants of the homeodomain ( L e Roux et al., 1993), demonstrate that the translocating property is due to the third helix of the homeodomain, a structure similar, if not identical, in a very large series of homeodomain and thus highly con- sewed during evolution. This suggests that translocation across the plasma membrane could be a conserved property for many homeodomains and could therefore be of real physiological significance.

Acknowledgments-We thank Dm. J.-P. Henry, M. Thiefliy, and L. Theodore for careful reading of the manuscript and invaluable advice. We also acknowledge Drs. A. Brunissen, G. Hui Bon Hoa, and M. Mon- not for help with some of the experiments.

REFERENCES 1. Beaumelle, B., Bensammar, L., and Bienveniie, A. (1992) J. Biol. Chern. 267,

2. Bloch-Gallego, E., Le Row, I., Joliot, A. H.. Volovitch, M., Henderson, C. E., 11525-11531

and Prochiantz, A. (1993) J. Cell Biol. 120, 485-492

4. Glick, B., and Schatz. C . (1991)Annu. Rev. Genet . 2 6 , 2 1 4 3. Gehring, W. J. (1987) Science 236.1245-1252

5. Holland, P. (1992) BioEssays 14,267-273 6. Hunt, P., and Krumlauf, R. (1992) Annu. Rev. Cell. Biol. 8,227-256 7. Jiang, J. X., Chung, L. A,, and London. E. (1991) J. Biol. Chem. 266,24003-

8. Joliot, A,, Pernelle, C., Deagostini-Bazin, H., and Prochiantz, A. (1991a) Proc.

9. Joliot. A,. Triller. A,, Volovitch. M., Pernelle, C.. and Prochiantz,A. (1991b) The

24010

Natl. Acad. Sci. U. S. A. 88, 1864-1868

10. Lafont, F., Rouget, M.. Triller, A,, Prochiantz, A., and Rousselet, A. (1992) New Biol. 3,1121-1134

11. Lemmon, M. A,, Flanagan, J. M., Hunt, J. H., Adair, B. D., Bormann. B. J., Development -114, 17-29

Dempsey, C. E., and Engelman, D. M. (1992a) J. Biol. Chem. 267, 7683- 7689

12. Lemmon, M.A., Flanagan, J. M., Treutlein, H. R., zhang, J., and Engelman. D.

13. Le Roux, I., Joliot, A. H., Bloch-Gallego, E., Prochiantz, A,, and Volovitch. M.

14. Maduke, M., and Roise, D. (1993) Science 260,364-367 15. Moskaug, J. 0.. Stenmark, H., and Olsnes, S . (1991) J. Biol. Chem. 266,

16. Moya, M., Qautry-Varsat, A,, Goud, B., Louvard, D., and Boquet, P. (1985) J.

17. Olsnes, S. . Moskaug, J. 0.. Stenmark, H., and Sandvig, K. (1988) %Rends

18. Papini, E., Rappuoli, R., Murgia, M., and Montecucco, C. (1993)J. Bwl. Chem.

19. Perez, F.. Joliot, A,. Bloch-Gallego, E., 2ahraoui.A.. Triller, A., and Prochiantz,

~~

M. (1992b) Biochemistry 31,12719-12725

(1993) Proc. Natl. Acad. Sci. U. S. A. 90,9120-9124

2652-2659

Cell Biol. 101, 548-559

Biochem. Sci. 13,348451

268, 1567-1574

20.

21.

22. 23. 24.

25.

Price, M., Lazzaro, D., Pohl, T., Mattei, M.-G., Riither, U., Olivo, J.-C.,

Qian. Y. Q., Billeter, M., Otting, G., Muller, M.. Gehring, W. J., and Wuthrich,

Rapoport, T. A. (1992) Science 258.931-936 Reisfeld. R. A., Lewis, V. J.. and Williams, D. E. (1962) Nature 196,281-283 Simeone, A., Gulisano, M., Acampora, D., Stornaiuolo, A,, Rambaldi, M., and

Vallette, E M.. Juin, P., Pelleschi, M., and Henry, J.-P. (1994) J. Biol. Chern. in

A. (1992) J. Cell Sci. 102,717-722

Duboule. D., and Di Lauro, R. (1992) Neuron 8,241-255

K. (1989) Cell 59,573-580

Boncinelli, E. (1992) EMBO J. 11,2541-2550

press

Documents

The Third Helix of the Antennapedia Homeodornain Translocates through Biological