From the UMR 5539 CNRS, Département
Biologie-Santé, Université Montpellier II, 34095 Montpellier, § U 476 INSERM, Faculté de
Médecine, 27 Boulevard Jean Moulin, 13385 Marseille, and
Laboratoire de Bioénergétique Cellulaire,
Département d'Ecophysiologie Végétale et
Microbiologie, CEA-Cadarache,
13107 Saint Paul lez Durance, France
Received for publication, September 17, 2002, and in revised form, February 26, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ricin is a heterodimeric plant toxin and the
prototype of type II ribosome-inactivating proteins. Its B-chain
is a lectin that enables cell binding. After endocytosis, the A-chain
translocates through the membrane of intracellular compartments
to reach the cytosol where its N-glycosidase activity
inactivates ribosomes, thereby arresting protein synthesis. We here
show that ricin possesses a functional lipase active site at the
interface between the two subunits. It involves residues from both
chains. Mutation to alanine of catalytic serine 221 on the A-chain
abolished ricin lipase activity. Moreover, this mutation slowed down
the A-chain translocation rate and inhibited toxicity by 35%. Lipase
activity is therefore required for efficient ricin A-chain
translocation and cytotoxicity. This conclusion was further
supported by structural examination of type II ribosome-inactivating
proteins that showed that this lipase site is present in toxic (ricin
and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I)
members of this family.
Ricin, isolated from seeds of the plant Ricinus
communis, is the prototype of type II ribosome-inactivating
proteins (RIPs)1 and one of
the most powerful toxins capable of killing animal cells. This 66-kDa
glycoprotein is composed of two chains (RTA and RTB) linked
via a disulfide bond. RTA is responsible for cytotoxicity. This N-glycosidase catalyzes the depurination of a specific
adenine on the 28 S ribosomal RNA, thereby inactivating protein
synthesis and leading to cell death. RTB is a lectin that recognizes
terminal galactose residues and is responsible for toxin binding to
cells (1).
X-ray structures of the heterodimer and the recombinant RTA subunit
(rRTA) have been solved at 2.5 and 2.3 Å, respectively (2, 3). These
studies described both the RTA N-glycosidase active site and
the RTB galactose binding pockets. rRTA lacks the glycans present on
native RTA but shows complete biological activity (1, 4).
Ricin has been used to study molecular mechanisms involved in
intracellular trafficking (5). After cell binding, ricin is endocytosed
and can be visualized in endosomes and the trans-Golgi network (6). Biochemical evidence indicates that it could be transported back to the endoplasmic reticulum (7). Ricin escape to the
cytosol has been reported to occur from endosomes (8), the Golgi
apparatus (9), and the endoplasmic reticulum (7). It is not known
whether the affinity of RTA for model membranes (10, 11) facilitates
its trans-membrane transport. This translocation step is
rate-limiting for cytotoxicity (12).
Ricin has been extensively studied for its potential use in cancer
treatments. Toward this objective, immunotoxins (ITs) were prepared by
attaching ricin to a monoclonal antibody (13). For clinical use and to
avoid nonspecific binding of the IT, B-chain lectin sites were
chemically inactivated. ITs containing such blocked ricin are usually
much more efficient in vivo than those prepared with RTA
alone (13). Similar data were obtained in vitro when lactose
was used to prevent RTB binding to cells, i.e., ricin-containing ITs were more toxic than ITs made from RTA (14). Hence, RTB involvement in ricin toxicity is not restricted to cell
binding mediated by its lectin sites (15). The basis of this
potentiation effect of RTB on RTA toxicity, even when RTB lectin
activity is ineffective, is not known.
Following our initial observation that ricin displays significant
homology to a lipase from another Euphorbiaceae (16), we recently
demonstrated that ricin shows lipolytic activity (17) and further
characterized its specificity toward neutral lipids (18). Such lipase
activity could facilitate RTA access to the cytosol by providing local
destabilization of the membrane (18) and might therefore be implicated
in the potentiation of RTA toxicity by RTB. The B-chain would thus
directly assist the A-chain during its translocation step as is the
case for another heterodimeric toxin, diphtheria toxin (19).
In this study, we localized a single canonical lipase catalytic triad
within ricin and localized at the junction between RTA and RTB. We
demonstrated the functionality of the ricin lipase site, which is
conserved in toxic but not in innocuous members of the type II RIP family.
Materials--
Ricin, CM- and Blue-Sepharose, and most chemicals
were obtained from Sigma. [35S]methionine
(Trans-label) was from ICN. Pure RTB (without any detectable RTA) was
purchased from Inland Labs (Austin, TX). RTA was purified from a
commercial preparation (Sigma) using chromatography on lactose-agarose
(8) to remove contaminating ricin. Transferrin was labeled with Cy5
using a labeling kit (Amersham Biosciences). Secondary antibodies for
immunofluorescence were from Nordic.
Mutagenesis--
The full-length RTA coding sequence (20) in pKK
223.3 (Pharmacia) was used as a template for PCR-based mutagenesis
(21). A 5'-mutagenic primer
GCAATTCAAGAGGCTAACCAAGGAGCC, in which the TCT
coding for Ser-221 was changed to the underlined GCT (Ala), was used
together with a 3'-primer overlapping the downstream plasmid-HindIII site to prepare a first PCR fragment. A
3'-mutagenic primer and a 5'-primer spanning the upstream
ClaI site enabled obtaining a second PCR fragment. These
overlapping fragments were then joined and amplified by a second PCR
using the outer primers. The resulting
ClaI-HindIII fragment was inserted into pKK
223.3-RTA for expression. A similar strategy was adopted to change
His-40 to Ala, using a 5'-mutagenic primer in which the CAT coding for His-40 was altered to GCT (Ala), together with a 3'-primer covering the
ClaI site. The upstream fragment was prepared using a
5'-primer covering the plasmid EcoRI site and a 3'-mutagenic
primer prior to the second round of PCR run to join the fragments.
PCR-amplified DNA was entirely sequenced.
Purification and RNA N-Glycosidase Assays of rRTA and rRTA
Mutants--
A 1.5-liter culture of transfected Escherichia
coli TG2 was grown at 30 °C. Expression was induced at an
A595 of ~0.7, using 1 mM
isopropylthiogalactoside. After 2.5 h at 30 °C, E. coli lysates were prepared by sonication, clarified by
centrifugation for 30 min at 25,000 × g, and dialyzed
against 10 mM sodium phosphate buffer, pH 6.8 (buffer A),
before loading onto a CM-Sepharose column. After washing with buffer A,
rRTA was eluted with a NaCl gradient of 0-1 M in buffer A
(20). A-chains were further purified by affinity chromatography on
Blue-Sepharose and assayed for N-glycosidase activity using
rabbit reticulocyte lysates (Promega) as indicated (8).
Association of rRTA with RTB--
Equimolecular amounts of rRTA
and plant RTB (20 µM in phosphate-buffered saline) were
mixed in the presence of 8 mM reduced glutathione. After
3 h at room temperature and overnight dialysis at 4 °C against
phosphate-buffered saline (4), the reconstituted control or mutated
ricins were filtered-sterilized and stored at 4 °C for up to 1 week.
Lipase and Toxicity Assays--
RTA, rRTA, RTB, native, control,
or mutant ricins (0-150 nmol) were added to 1 ml of 100 mM
Tris-HCl, pH 8.0, containing 2 mM
5,5'-dithiobis(2-nitrobenzoic acid) and 2 mM of
2,3-dimercapto-1-propanol tributyrate (BAL-TC4; Aldrich).
After 30 min at 37 °C, 2 ml of acetone were added before measuring
the A412 as described previously (18). Specific
lipolytic activities were expressed as millikatal/mol. A katal
is the amount of enzyme which transforms 1 mol of substrate per second.
Bovine serum albumin, used as control, did not show any lipolytic
activity. Peripheral blood mononuclear cells were purified from human
blood using Ficoll-Paque plus (Amersham Biosciences), and monocytes
were allowed to adhere overnight to tissue culture plates before
washing and adding ricins. After 24 h, the medium (RPMI
complemented with 10% human AB+ serum) was replaced for
4 h with Dulbecco's modified Eagle's medium without methionine,
supplemented with [35S]methionine. This medium was then
aspirated. Cell proteins were precipitated with trichloroacetic acid
and collected by filtration before scintillation counting (4).
Experiments were performed in triplicate and repeated three times.
Errors are expressed as S.E.
Kinetics of Protein Synthesis Inactivation by Control or Mutated
Ricin--
A431 cells in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum were seeded into 96-well plates
(24,000 cells/well), whereas monocytes were used at 40-60,000
cells/well. Ricins were added, and a 1-h (A431) or 2-h (monocytes)
pulse incorporation of [35S]methionine in Dulbecco's
modified Eagle's medium without methionine was performed at various
times after the start of intoxication (4).
Internalization Studies--
Ricins were radiolabeled with
125I to monitor their endocytosis efficiency at 37 °C by
mouse BW 5147 lymphocytes, using 0.1 M lactose to displace
plasma membrane-bound ricin molecules (4). For fluorescence microscopy,
A431 cells were labeled for 40 min at 37 °C with 30 nM
ricin and 200 nM transferrin-Cy5 in Dulbecco's modified
Eagle's medium supplemented with 0.2 mg/ml bovine serum albumin before
lactose scraping. Cells were further washed, fixed, permeabilized with
0.015% saponin, and labeled with antibodies. The rabbit anti-ricin
antibodies (Sigma) were revealed using TRITC-labeled swine anti-rabbit
IgG; sheep anti-TGN46 (Serotec) and the H4A3 anti-Lamp-1 monoclonal
antibody (Iowa Developmental Studies Hybridoma Bank) were detected
using fluorescein isothiocyanate-conjugated donkey anti-sheep and goat
anti-mouse antibodies, respectively. After mounting, cells were
examined under a Leica confocal microscope (22).
Structure Analysis--
Structures of ricin (PDB code 2AAI),
neutral Pseudomonas lipase (5TGL), abrin (1ABR) mistletoe
lectin I (1CE7), and ebulin (1HWO) were analyzed using the graphic
program O (23) running on a Silicon Graphics workstation.
We first tried to identify the ricin chain responsible for
heterodimer lipase activity. Purified chains showed lipase activities below 18% as compared with native ricin (Fig.
1A). We concluded that the
lipase site is found in the heterodimer only.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Lipolytic activity of ricin chains, native,
control, and mutated (rRTA221A) ricin. Proteins were assayed for
lipase activity at 37 °C using BAL-TC4 as substrate.
Activities are expressed as femtokatal. A, native
ricin, RTA, rRTA, and RTB. B, control (rRTA-RTB) and
mutant (rRTA221A-RTB) ricin.
Localization of a Putative Catalytic Triad--
All lipolytic
enzymes investigated so far vary widely in size and amino acid
sequence. However, most of them belong to the /
hydrolase
superfamily in which the catalytic machinery consists of a nucleophile,
an acid, and a histidine residue (the catalytic triad). Nucleophilic
serine is located at the center of an extremely sharp turn between a
-strand and an
-helix. The sharpness of the turn results in phi
and psy angles that lie in an unfavorable region of the Ramachandran
plot (24). A specific feature of lipases, as compared with canonical
/
hydrolases, is that the active site is inaccessible to
substrate in solution. In the presence of lipids, unmasking the active
site generates the hydrophobic substrate binding site. This
conformational change generates the so-called oxyanion hole where the
transition state of the reaction can be stabilized via hydrogen bonds
with two main-chain nitrogens (25).
Ricin lipase activity is more active (~5-fold) on neutral lipids,
such as triglycerides (18), than on glycerophospholipids (17). We
therefore investigated the crystal structure of ricin to find common
structural features with /
hydrolases, using the typical neutral
lipase of Pseudomonas as reference (26). This led us to find
only one putative serine hydrolase catalytic triad (RTA-Ser-221,
RTA-His-40, and RTB-Asp-94). Nucleophilic RTA-Ser-221 is located at the
beginning of a small turn between an
-helix (from 202 to 220) and a
-strand (from 227 to 234) (Fig. 2).
Nevertheless, as opposed to
/
hydrolases, the catalytic serine
lies in a favorable region of the Ramachandran plot. RTA-Ser-221 is
hydrogen-bonded to RTA-His-40, which in turn interacts with Asp-94 of
the RTB subunit. The distances between RTA-Ser-221 o
and RTA-His-40
N
2 and between RTA-His-40 N
1 and RTB-Asp-94 o
are 3.2 and 2.8 Å, respectively. These values are almost identical to those measured
in our reference lipase active site (3.1 and 2.8 Å). Hence, the
relative orientation of side chains involved in both catalytic triads
are strikingly similar, whereas ricin and lipase folding is completely
different (Fig. 3, A and
B). RTA-Ser-221 is located at the bottom of a hydrophobic
groove, which might correspond to the binding site of substrate acyl
chains. No attempt was made here to model a putative conformational
change similar to lipase activation. The putative substrate binding
site is located at the interface between ricin subunits (Fig.
3B). Such localization explains why the heterodimer only
shows significant lipase activity (Fig. 1A).
|
|
The Putative Lipase Catalytic Site Is Functional--
Among the
three residues of the putative catalytic triad, two of them, RTA-His-40
and RTB-Asp-94, directly interact and are involved in A/B polar
interactions (27). Accordingly, when rRTA-His-40 was changed to Ala and
produced in E. coli, the resulting rRTA40A associated very
poorly with RTB as compared with control rRTA (Fig.
4). Not surprisingly, this rRTA40A-RTB
preparation, containing a large proportion (~80%) of isolated
chains, did not show significant lipase activity (data not shown).
Because this mutation prevented heterodimer formation, and therefore
building of the lipase site, it was not possible to deduce from this
lack of lipase activity any information regarding the role of
RTA-His-40 in lipase activity. RTA-His-40 and RTB-Asp-94 only interact
with each other across the A/B interface (27). An RTB-Asp-94 mutation
would thus likely destabilize the heterodimer exactly as the rRTA-H40A
change, and so we did not prepare such a mutant.
|
The third residue of the ricin lipase site, RTA-Ser-221, is not implicated in A/B interaction and was potentially catalytic. It was therefore the most appropriate for a point mutation in order to study the functionality of this site. We replaced rRTA-Ser-221 by an alanine. The rRTA221A mutant was produced in E. coli. It could form the heterodimer as efficiently as control rRTA (Fig. 4). Again, this was not surprising because Ser-221 does not interact with any B-chain residue (27). RTA-Ser-221 is located far away from the N-glycosidase active site cleft (around 25 Å). Nevertheless, several amino acids outside this cleft can, albeit indirectly, participate in N-glycosidase catalysis (28). Hence, it was important to check whether the S221A mutation affected this activity. We therefore tested the abilities of RTA, rRTA, rRTA40A, and rRTA221A to inhibit cell-free protein synthesis in reticulocyte lysates. No significant difference in N-glycosidase activity was found (not shown). We therefore concluded that the rRTA221A mutation did not affect the ribosome-inactivating activity of the protein.
We then compared the properties of these control (rRTA-RTB) and mutant (rRTA221A-RTB) ricins. Wild-type (RTA-RTB) and control (rRTA-RTB) ricins displayed indistinguishable lipase activities: ~3.3 millikatal/mol (Fig. 1, A and B) on BAL-TC4. This demonstrates that the lipase active site of ricin is correctly assembled upon formation of control ricin. The S221A mutation on the A-chain decreased heterodimer lipase activity to non-significant levels, corresponding to a 94 ± 5% inhibition compared with control ricin (Fig. 1B). This demonstrates the catalytic role of RTA-Ser-221 in lipid hydrolysis and is consistent with the 89% inhibition obtained using a serine hydrolase inhibitor, i.e. E600 (18). The lipase site predicted by structural analysis is therefore functional in ricin and unique within the molecule.
Lipase Activity Does Not Affect Ricin Intracellular
Routing--
We then assessed the involvement of ricin lipolytic
activity in its intracellular transport using rRTA221-RTB. Cell binding and global endocytosis efficiency were monitored via the
uptake of 125I-ricin by mouse lymphocytes (4). This assay
did not reveal any difference between control and mutant ricin (not
shown). Ricin intracellular routing was examined by immunofluorescence
confocal microscopy using fluorescent transferrin, TGN46, and Lamp-1 as markers of early endosomes (22), the trans-Golgi network
(29), late endosomes and lysosomes (30), respectively. Both control and
mutant ricins were efficiently endocytosed by A431 cells (Fig. 5). Consistent with numerous studies of
ricin uptake by several cell types (8, 31, 32), they localized
essentially to elements of the endo/lysosomal pathway, together with
transferrin and Lamp-1. In agreement with previous data demonstrating
the transport of a small fraction of endocytosed ricin to the
trans-Golgi network (31), weak colocalization of ricins with
TGN46 was observed. Control and mutant ricins (shown in Fig. 5 for
control and S221A, not shown for H40A) displayed the same intracellular
pathway when studied through this assay. We concluded that lipolytic
activity is not involved in ricin intracellular trafficking.
|
Lipase Activity Is Implicated in Ricin Cytotoxicity and
Translocation--
We then tested whether mutations within a ricin
lipase site would affect toxicity, using monocytes from human
peripheral blood as target cells. rRTA221A-RTB with an IC50
of 3 pM was ~35% less toxic than control ricin
(rRTA-RTB), which showed an IC50 of 2 pM for
these cells (Fig. 6). rRTA40A-RTB was the
least toxic molecule (IC50 of l0 pM), probably
because of the weak A/B interaction for this mutant.
|
Ricin translocation was examined using kinetics of protein synthesis
inactivation. This assay enables translocation assessment on intact
cells (4, 12). It therefore provides data that are not restricted to
the observation of ricin translocation from a specific organelle. The
slope of the plot log (protein synthesis) versus time,
measured after the initial dose-dependent lag, is directly
proportional to the RTA translocation rate (12). As shown in Fig.
7A for A431 cells and Fig.
7B for monocytes, mutant ricin (rRTA221A-RTB) killed cells
more slowly than the control toxin. Because the mutation did not affect
rRTA N-glycosidase activity or intracellular routing, the
difference in the cell-killing rate was because of a lower
translocation efficiency of mutant rRTA. When translocation rates were
calculated from the best slope of these plots, whatever the cell line,
rRTA221A translocation efficiency was only 64 ± 5% (mean ± S.E.; n = 7; p < 0.001) compared with
that of rRTA. This result demonstrates that ricin lipase activity is
implicated in toxicity, presumably during the translocation step.
Although the effect of the RTA221A mutation on cytotoxicity and
translocation may appear moderate, it should be noted that conjugates
made only from RTA are already toxic, showing that, once bound to
cells, RTA is able to translocate quite efficiently on its own.
Nevertheless, conjugates prepared using blocked ricin are usually more
efficient (13). Second, ricin lipase activity is low (18). We therefore
propose that the lipase activity of the heterodimer is likely
responsible for this potentiation effect of the B-chain even when
lectin sites are inactivated (14). The B-chain therefore assists and
facilitates A-chain translocation within the animal cell.
Interestingly, compartments where ricin translocation was reported to
take place, such as late endosomes (4) and the endoplasmic reticulum
(7), are specifically enriched in triglycerides compared with the
plasma membrane (33, 34). Because these lipid species are the preferred
substrate for ricin lipase activity (18), potentiation of A-chain
translocation likely results from local destabilization of the membrane
of specific, triglycerides-rich intracellular compartments by moderate
lipase activity of the heterodimer.
|
Mutation of RTA-His-40 did not give rise to a mutant suitable for studying the role of this residue in lipase activity, because the mutation prevented the A/B association required to generate the site. Hence, suppression of a single point of A/B interaction among the 15 pairs of residues enabling stabilization of the heterodimer (in addition to the interchain disulfide) (27) led to a dramatic heterodimer dissociation. Because the heterodimer does not show any RNA N-glycosidase activity, the A-chain has to be released at a late stage of the intoxication process, presumably following reduction of the interchain disulfide (35). The A/B interaction, which showed a Kd in the micromolar range (36), is therefore easily reversible; it was not very surprising that removing one point of interaction via the RTA40A mutation induced massive dissociation.
The ricin lipase site is, to our knowledge, the first ever reported to
be constituted by residues from distinct subunits. This could be a
feature of plant lipases, and ricin is the first one whose structure is
available. Because ricin is the prototype of type II RIPs, it was
interesting to examine whether the lipase site was also present in
other members of this family whose structures are available. Abrin, a
toxin structurally and functionally related to ricin (37), also
displays a potential lipase catalytic site made of Ser-208 (A-chain),
His-34 (A-chain), and Asp-99 (B-chain) (Fig. 3C). Structural
analysis clearly showed (Fig. 3) that the lipolytic site is not
conserved in the two barely toxic type II RIPs, mistletoe lectin I (38)
and ebulin 1 (39). Hence, there is a correlation between the presence
of a canonical lipase site and toxicity in type II RIPs. This finding
further indicates that lipolytic activity likely plays an important
role in their cytotoxicity.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a grant from the French government.
** Supported by the Conseil Général des Bouches du Rhône.
To whom correspondence should be addressed: UMR 5539 CNRS, Case
107, Université Montpellier II, 34095 Montpellier Cedex 05, France. Tel.: 33-467-14-33-98; Fax: 33-467-14-42-86; E-mail:
beaumel@univ-montp2.fr.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M209516200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RIP, ribosome-inactivating protein; RTA, ricin toxin A-chain; RTB, RT B-chain; rRTA, recombinant RTA; ITs, immunotoxins; TRITC, tetramethylrhodamine isothiocyanate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Lord, J. M.,
Roberts, L. M.,
and Robertus, J. D.
(1994)
FASEB J.
8,
201-208 |
2. |
Montfort, W.,
Villafranca, J. E.,
Monzingo, A. F.,
Ernst, S. R.,
Katzin, B.,
Rutenber, E.,
Xuong, N. H.,
Hamlin, R.,
and Robertus, J. D.
(1987)
J. Biol. Chem.
262,
5398-5403 |
3. |
Mlsna, D.,
Monzingo, A. F.,
Katzin, B. J.,
Ernst, S.,
and Robertus, J. D.
(1993)
Protein Sci.
2,
429-435 |
4. |
Beaumelle, B.,
Taupiac, M. P.,
Lord, J. M.,
and Roberts, L. M.
(1997)
J. Biol. Chem.
272,
22097-22102 |
5. |
Sandvig, K.,
and van Deurs, B.
(2000)
EMBO J.
19,
5943-5950 |
6. | van Deurs, B., Tonnessen, T. I., Petersen, O. W., Sandvig, K., and Olsnes, S. (1986) J. Cell Biol. 102, 37-47[Abstract] |
7. |
Wesche, J.,
Rapak, A.,
and Olsnes, S.
(1999)
J. Biol. Chem.
274,
34443-34449 |
8. |
Beaumelle, B.,
Alami, M.,
and Hopkins, C. R.
(1993)
J. Biol. Chem.
268,
23661-23669 |
9. |
Bilge, A.,
Warner, C. V.,
and Press, O. W.
(1995)
J. Biol. Chem.
270,
23720-23725 |
10. | Utsumi, T., Ide, A., and Funatsu, G. (1989) FEBS Lett. 242, 255-258[CrossRef][Medline] [Order article via Infotrieve] |
11. | Day, P. J., Pinheiro, T. J., Roberts, L. M., and Lord, J. M. (2002) Biochemistry 41, 2836-2843[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Hudson, T. H.,
and Neville, D. M.
(1987)
J. Biol. Chem.
262,
16484-16494 |
13. | Kreitman, R. J. (1999) Curr. Opin. Immunol. 11, 570-578[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Gregg, E. O.,
Bridges, S. H.,
Youle, R. J.,
Longo, D. L.,
Houston, L. L.,
Glennie, M. J.,
Stevenson, F. K.,
and Green, I.
(1987)
J. Immunol.
138,
4502-4508 |
15. | Frankel, A. E., Fu, T., Burbage, C., Chandler, J., Willingham, M. C., and Tagge, E. P. (1997) Leukemia 11, 22-30[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Moulin, A.,
Teissere, M.,
Bernard, C.,
and Pieroni, G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11328-11332 |
17. | Helmy, M., Lombard, S., and Piéroni, G. (1999) Biochem. Biophys. Res. Commun. 258, 252-255[CrossRef][Medline] [Order article via Infotrieve] |
18. | Lombard, S., Helmy, M. E., and Pieroni, G. (2001) Biochem. J. 358, 773-781[CrossRef][Medline] [Order article via Infotrieve] |
19. | Falnes, P. O., and Sandvig, K. (2000) Curr. Opin. Cell Biol. 12, 407-413[CrossRef][Medline] [Order article via Infotrieve] |
20. | Chaddock, J. A., and Roberts, L. M. (1993) Protein Eng. 6, 425-431[Abstract] |
21. | Landt, O., Grunert, H.-P., and Haln, U. (1990) Gene 96, 125-128[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Alami, M.,
Taupiac, M. P.,
Reggio, H.,
Bienvenue, A.,
and Beaumelle, B.
(1998)
Mol. Biol. Cell
9,
387-402 |
23. | Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
24. | Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., and Schrag, J. (1992) Protein Eng. 5, 197-211[Abstract] |
25. | Jaeger, K. E., Ransac, S., Dijkstra, B. W., Colson, C., van Heuvel, M., and Misset, O. (1994) FEMS Microbiol. Rev. 15, 29-63[CrossRef][Medline] [Order article via Infotrieve] |
26. | Schrag, J. D., Li, Y., Cygler, M., Lang, D., Burgdorf, T., Hecht, H. J., Schmid, R., Schomburg, D., Rydel, T. J., Oliver, J. D., Strickland, L. C., Dunaway, C. M., Larson, S. B., Day, J., and McPherson, A. (1997) Structure 5, 187-202[Medline] [Order article via Infotrieve] |
27. | Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269[Medline] [Order article via Infotrieve] |
28. | Kitaoka, Y. (1998) Eur. J. Biochem. 257, 255-262[Abstract] |
29. |
Rohrer, J.,
and Kornfeld, R.
(2001)
Mol. Biol. Cell
12,
1623-1631 |
30. | Gruenberg, J., and Maxfield, F. R. (1995) Curr. Opin. Cell Biol. 7, 552-563[CrossRef][Medline] [Order article via Infotrieve] |
31. | van Deurs, B., Sandvig, K., Peterson, O. W., Olsnes, S., Simons, K., and Griffiths, G. (1988) J. Cell Biol. 106, 253-267[Abstract] |
32. | van Deurs, B., Hansen, S. H., Petersen, O. W., Melby, E. L., and Sandvig, K. (1990) Eur. J. Cell Biol. 51, 96-109[Medline] [Order article via Infotrieve] |
33. | Kobayashi, T., Stang, E., Fang, K. S., de Moerloose, P., Parton, R. G., and Gruenberg, J. (1998) Nature 392, 193-197[CrossRef][Medline] [Order article via Infotrieve] |
34. | Hussain, M. M. (2000) Atherosclerosis 148, 1-15[CrossRef][Medline] [Order article via Infotrieve] |
35. | Olsnes, S., Sandvig, K., Refsnes, K., and Pihl, A. (1976) J. Biol. Chem. 251, 3985-3992[Abstract] |
36. |
Lewis, M. S.,
and Youle, R. J.
(1986)
J. Biol. Chem.
261,
11571-11577 |
37. | Tahirov, T. H., Lu, T. H., Liaw, Y. C., Chen, Y. L., and Lin, J. Y. (1995) J. Mol. Biol. 250, 354-367[CrossRef][Medline] [Order article via Infotrieve] |
38. | Eschenburg, S., Krauspenhaar, R., Mikhailov, A., Stoeva, S., Betzel, C., and Voelter, W. (1998) Biochem. Biophys. Res. Commun. 247, 367-372[CrossRef][Medline] [Order article via Infotrieve] |
39. | Pascal, J., Day, P. J., Monzingo, A. F., Ernst, S. R., Robertus, J. D., Iglesias, R., Perez, Y., Ferreras, J. M., Citores, L., and Girbes, T. (2001) Proteins 43, 319-326[CrossRef][Medline] [Order article via Infotrieve] |
40. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |