The Prodomain of a Secreted Hydrophobic Mini-protein Facilitates Its Export from the Endoplasmic Reticulum by Hitchhiking on Sorting Receptors*
Silvestro G. Conticello
,
Noga D. Kowalsman
,
Christian Jacobsen
,
Guennady Yudkovsky ¶,
Kazuki Sato ||,
Zvulun Elazar
,
Claus Munck Petersen
,
Ami Aronheim ¶ and
Mike Fainzilber
**
From the
Department of Biological Chemistry,
Weizmann Institute of Science, 76100 Rehovot, Israel, the
Department of Medical Biochemistry, University
of Aarhus, DK-8000, Denmark, the ¶Department of
Molecular Genetics, Rappaport Faculty of Medicine, Technion, 31096 Haifa,
Israel, and the ||Fukuoka Women's University,
Kasumigaoka, Higashi-ku, Fukuoka 813-8529, Japan
Received for publication, April 2, 2003
, and in revised form, May 16, 2003.
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ABSTRACT
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Misfolded secretory proteins are retained in the endoplasmic reticulum (ER)
by quality control mechanisms targeted to exposed hydrophobic surfaces.
Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon
folding to their bioactive structures. How then can such secreted
mini-proteins traverse the secretory pathway? Here we show that secretion of
the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain,
which enhances TxVI export from the ER. The propeptide domain interacts with
sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI
interaction occurs in the ER, and sortilin facilitates export of TxVI from the
ER to the Golgi. Thus, the prodomain in a secreted hydrophobic protein acts as
a tag that can facilitate its ER export by a hitchhiking mechanism.
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INTRODUCTION
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Biosynthesis and folding of secreted proteins are regulated by quality
control mechanisms targeted to exposed hydrophobic surfaces or free thiols on
misfolded proteins, thus retaining them in the
ER1
(1,
2). Misfolded proteins that
escape ER quality control can also be directed to vacuolar degradation from
the trans-Golgi network (TGN) or returned via retrograde transport to the ER
(3). Paradoxically, certain
conotoxins expose extensive hydrophobic surfaces upon folding to their stable
bioactive structures (Fig.
1A and Refs.
4 and
5), suggesting the existence of
mechanisms for efficient secretion of hydrophobic mature domains. Conotoxin
precursors consist of a signal peptide, a propeptide domain, and the mature
toxin domain. An early suggestion that the propeptide domain might act as an
intramolecular chaperone (6)
was not supported by in vitro folding experiments on a hydrophilic
conopeptide (7). Nonetheless,
the high degree of propeptide conservation in hydrophobic conotoxins
(Fig. 1B) indicated
that this domain might be required in vivo for efficient secretion.
We used the extremely hydrophobic conotoxin-TxVI
(8) to examine this question.
Here we show that secretion of TxVI is strongly dependent on its propeptide
domain, which enhances TxVI export from the endoplasmic reticulum (ER) by
hitchhiking on sorting receptors from the sortilin Vps10p domain family.

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FIG. 1. Enhancement of TxVI secretion by its propeptide. A:
left, Conus textile, a cone snail with a remarkably high proportion
of hydrophobic conopeptides in its venom duct; Right, space filling
representation of the NMR structure of conotoxin-TxVI. Hydrophobic surfaces
are indicated in white, acidic in red, and others in
blue. B, sequence alignment by domains of hydrophobic conopeptides.
C, pulse-chase analysis of Fc-tagged constructs of TxVI with and
without the propeptide (FL and P, respectively).
Representative gels are shown for TxVI-Fc protein in the medium (m)
and in the cells (c) at given times (hours (hr)) after a
30-min pulse with [35S]Cys-Met in transiently transfected COS7
cells. D, plot of secretion kinetics for the different constructs,
average ± S.D. from four independent experiments (FL, solid
circle; P, hollow square). Equivalent amounts of
transfected cells were used. Secretion levels are expressed as percentage of
the total de novo synthesized protein measured at the end of the
pulse. Relative de novo synthesis levels for both constructs at the
end of the pulse are shown in the inset bar graph, as percentage of
the synthesized P protein.
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EXPERIMENTAL PROCEDURES
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PlasmidsAn FL-Fc construct comprised of the open reading
frame of TxVI (GenBankTM accession number AF193261
[GenBank]
) fused at its
C-terminal to the Fc domain of human IgG was cloned into pCDNA3. A propeptide
deletion (
P) was prepared by ligating oligonucleotides encoding the
signal peptide to a PCR product encoding the mature toxin domain fused with
the Fc. A GFP fusion (FL-GFP) was constructed by excision of the Fc domain
followed by ligation of EGFP (Clontech, Palo Alto, CA). A GFP construct
targeted to the ER (Sig-GFP) was made by replacing the TxVI sequence of FL-GFP
with a signal sequence derived from the p75 neurotrophin receptor.
Cell Culture, Transfection, and Pulse ChaseCOS7 cell lines
were maintained in DMEM with 10% fetal calf serum (Biological Industries, Beit
Haemek, Israel), and transfected with FuGENE-II (Roche Molecular
Biochemicals). Pulse-chase experiments were carried out 2448 h
post-transfection. Cells were transferred to serum-free DMEM deficient in
cysteine and methionine (Sigma), and pulsed in medium supplemented with
[35S]Cys-Met (0.1 mCi/ml) (Amersham Biosciences). Cells were chased
in full medium. Cell lysates were in 10 mM Tris, pH 8, 150
mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1 mM orthovanadate and proteinase
inhibitors (Merck).
Gel Analysis and Quantitation35S-Labeled
proteins were separated on 10 or 12% SDS-polyacrylamide gels and exposed on a
BAS-2500 (Fuji) phosphorimager screen. Results were analyzed and quantified
using Image Gauge 3.41 software (Fuji).
Protein Interaction ScreensThe RRS system selects for
protein interactions that rescue a yeast strain in which the endogenous Ras
signaling pathway is repressed at 36 °C. Yeast strains, plasmids, and
screening protocols for RRS were as described previously
(9). Baits were prepared by
cloning the propeptide domains of TxVI, PnIB, or BDNF in pADH-Ras, and
proTxVI-ADH-Ras was used to screen a rat pituitary cell line cDNA library.
Enzymatic, Antibody-based, or Protein Interaction Assays
endoglycosidase-H (Endo-H) (Roche Molecular Biochemicals) digests were carried
out as described previously
(10). Fc constructs were
purified over protein A-Agarose (Roche Molecular Biochemicals) according to
the manufacturer's instructions. A rabbit anti-sortilin antibody
(11) was used at 5 mg/ml for
precipitation and at 1:1500 dilution for Western blotting. Surface plasmon
resonance measurements were performed on BIAcore 2000 (Biacore) equipped with
CM5 sensor chips maintained at 20 °C under a continuous flow of
HEPES-buffered saline or MES buffers at the indicated pH
(12).
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RESULTS AND DISCUSSION
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Full-length and deletion constructs of TxVI were tagged C-terminally with
the Fc domain of IgG. This relatively large tag was deliberately chosen to
avoid the possibility of degradation of extremely short propeptide deletion
products (13). Equivalent
expression efficacy of the tagged proteins was verified by in vitro
translation, followed by metabolic labeling and pulse-chase experiments in
transfected COS cells. Labeled TxVI-Fc was easily detectable in the medium of
cells transfected with the full-length construct and accumulated at a linear
rate for 8 h (Fig.
1C), thereby establishing that the capacity to
efficiently secrete domains with high surface hydrophobicity is not restricted
to Conus venom ducts. The intracellular expression level of the
proprotein deletion was approximately half that of the full-length protein,
and it was secreted at a 67-fold slower rate than that observed for the
full-length product (Fig. 1, C and
D), resulting in an order of magnitude difference in the
amount of secreted TxVI. In similar experiments carried out with the
extracellular domain of the p75 neurotrophin receptor, which is composed of
four hydrophilic cysteine-rich domains, fusion with the TxVI-pro sequence did
not significantly enhance secretion of the construct from transfected cells
(data not shown).
To clarify the role of the pro domain in enhancing TxVI secretion, we
conducted an RRS screen for propeptide-interacting proteins and obtained five
independent clones corresponding to two overlapping segments of the lumenal
domain of sortilin (Fig.
2A). Sortilin is a member of the Vps10p domain family
(11,
14), members of which are
thought to be involved in TGN-endosome sorting
(15,
16). The sortilin-pro-TxVI
interaction is specific, as evidenced by a lack of interaction between
sortilin and the propeptide domains of two small cysteine-rich hydrophilic
proteins (Fig. 2B) and
by coimmunoprecipitation of sortilin with pro-TxVI from transfected mammalian
cells (Fig. 2C). Only
1% of the total cellular sortilin was co-precipitated with TxVI
(Fig. 2C), suggesting
that sortilin-TxVI interaction in the cell is spatially or temporally
restricted. Testing of a number of propeptide deletion constructs localized
the sortilin-binding domain to the N-terminal region of the propeptide
(Fig. 2D). Further
verification of the sortilin-pro-TxVI interaction was obtained by surface
plasmon resonance, demonstrating concentration dependent binding with an
apparent Kd of 1 µM and
stoichiometry of 0.2 mol of peptide bound per mol of sortilin immobilized
(Fig. 2E). Since less
than half of the sortilin is likely to be immobilized on the chip in an
orientation favorable for peptide binding, the latter value suggests binding
of the propeptide to a single site on sortilin. Binding is lost upon reduction
of sortilin (Fig. 2F).
The endogenous sortilin propeptide is known to bind to the lumenal domain of
mature sortilin, and its cleavage and dissociation activates certain functions
of sortilin (12). We therefore
tested for binding of TxVI propeptide in the presence of sortilin propeptide
and observed binding of the TxVI propeptide superimposed onto the binding
curve for sortilin propeptide, indicating that the binding sites for these two
ligands are distinct (Fig.
2G). Finally, we tested the TxVI propeptide for binding
to SorLA, another member of the Vps10p family
(17). Both full-length SorLA
and its isolated Vps10p domain bind TxVI propeptide, whereas megalin (an low
density lipoprotein domain-containing receptor that lacks the Vps10p domain)
does not (Fig.
2H).

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FIG. 2. The propeptide domain of TxVI interacts with the Vps10p domain of
sortilin family receptors. A, schematic showing the position of
the two cloned interactors on the full-length sortilin sequence (sortilin
amino acid positions 91353 and 123353, respectively).
B, RRS interaction between a full-length sortilin clone and the
propeptides of TxVI, -conotoxin-PnIB, or brain-derived neurotrophic
factor (BDNF). Triplicate yeast colonies are shown, grown at
permissive (24 °C) and non-permissive (36 °C) temperatures. The robust
colonies observed at 36 °C for TxVI-transfected yeast reveal the
interaction with sortilin. C, co-precipitation of sortilin with
TxVI-Fc following co-transfection in COS cells; the controls are
co-transfections with plasmids encoding GFP or Fc alone. The rightmost
lane shows total sortilin in 1% of the amount of cell lysate used for the
co-precipitation. D, interaction between sortilin and the indicated
deletion constructs of the TxVI propeptide, as revealed by growth of RRS yeast
colonies at 36 °C. E, concentration-dependent binding of the TxVI
propeptide to sortilin immobilized on a Biacore chip; the numbers
indicate TxVI propeptide concentration in micromolar. F, lack of
binding of pro-TxVI to sortilin that was reduced before immobilization.
G, binding of the TxVI propeptide to sortilin in the presence of
excess sortilin propeptide, demonstrating that the propeptide of sortilin does
not inhibit binding of the TxVI propeptide. H, TxVI propeptide binds
to the Vps10p domain (residues 1731) of sorLA with a response
indistinguishable from that observed upon binding to full-length (residues
12107) sorLA. Megalin, a sorLA homolog that lacks the Vps10p domain,
does not bind the TxVI propeptide.
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Since sortilin is thought to be involved in TGN-endosome trafficking, how
might it influence TxVI evasion of quality control mechanisms in the ER? One
possibility would be an interaction between sortilin and TxVI commencing in
the ER, followed by joint transport to the Golgi. Indeed localization of
sortilin binding to the N-terminal region of the TxVI prodomain, and the
ability of the TxVI prodomain to bind sortilin before cleavage of the sortilin
propeptide, both support such a scenario. Co-precipitates of TxVI-Fc and
sortilin from pulse-labeled transfected COS cells were therefore subjected to
Endo-H digest to discriminate between ER versus Golgi forms of the
proteins. The labeled sortilin band that co-precipitated with the TxVI-Fc was
clearly Endo-H-sensitive (Fig.
3A), confirming that the interaction between the two
proteins occurs in the ER. Biacore experiments
(Fig. 3B) then
revealed that the prodomain-sortilin interaction was pH-sensitive, with
affinity decreasing from pH 7.4 (ER) to pH 6.2 (Golgi), with a complete loss
of the interaction at pH 5.5 (secretory granules). Endo-H treatment of cell
lysates from pulse-chase experiments showed a higher efficiency of ER export
for full-length TxVI-Fc, with
6% of labeled full-length TxVI in the Golgi
after 1-h chase, compared with less than 1% for the prodeletion construct
(Fig. 3C). We then
proceeded to examine the rate of ER to Golgi trafficking of the TxVI-Fc
protein with and without co-transfected sortilin. Co-transfection of sortilin
enhanced ER export of TxVI, and the fraction of the protein in the Golgi at
later time points in the chase was increased from 6 to 8% at steady state in
control to 12% with additional sortilin
(Fig. 3D). In
complementary experiments sortilin was co-transfected with TxVI-GFP or with
signal peptide-GFP, and the complex was immunoprecipitated with anti-sortilin.
This allowed direct examination of ER-Golgi transport of sortilin by Endo-H
digestion, revealing that presence of the toxin markedly slows ER export of
sortilin (Fig. 3E).
Taken together, these data show that the sortilin-pro-TxVI interaction occurs
in the ER and that sortilin enhances ER export of the TxVI protein to the
Golgi.

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FIG. 3. Sortilin interaction with the TxVI propeptide enhances ER export.
A, Endo-H digest of sortilin from [35S]Cys-Met
pulse-chased transfected COS7 cells (10-min pulse-chase times indicated in
hours (hr)). The left panels show sortilin co-precipitated
in pull-downs of TxVI-Fc, while the right panel shows direct
immunopreciptation of sortilin. Since all the sortilin co-precipitated with
TxVI is Endo-H-sensitive, the interaction occurs in the ER. B,
Biacore analyses of TxVI propeptide binding to sortilin at three different pH
values, approximating ER (pH 7.4), Golgi (pH 6.2), and secretory granules (pH
5.5). C, plot showing ER export of TxVI-Fc with and without the
propeptide (FL, solid circle; P, hollow square).
Transfected cells were pulsed for 10 min with [35S]Cys-Met and
chased for the indicated times. D, ER export of TxVI-Fc in the
presence (solid circles) or in the absence (hollow circles)
of co-transfected sortilin. Values in C and D are expressed
as percentage of Endo-H-resistant TxVI-Fc from the total and represent average
± S.D. from three independent experiments. E, ER export of
sortilin in the presence of FL-TxVI-GFP (solid squares) or GFP with
the signal peptide of TxVI (hollow squares). Data are shown as
percentage of Endo-H-resistant sortilin from the total and represent average
± S.D. from three independent experiments. Statistical significance: *
indicates p < 0.05; ** indicates p < 0.01.
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Recently it has become apparent that ER export is a process regulated by
different signals and facilitating molecules
(18,
19). We have shown that the
prodomain of conotoxin-TxVI acts as a facilitator of ER export and enhances
secretion of this hydrophobic mini-protein. Intriguingly, the sortilin-binding
site in the prodomain is close to its N terminus, i.e. within
precisely those residues that are the first to enter the ER lumen during
translation of TxVI. The prodomain-sortilin interaction occurs in the ER; and
the TxVI propeptide binds a site on sortilin that is not masked by the
sortilin propeptide; thus, the interaction occurs before sortilin is activated
in the Golgi by truncation of its own propeptide. These data and the effect of
the proteins on each other's ER export kinetics fit a scenario whereby the
interaction essentially occurs while both proteins are co-maturing. The
slowing of sortilin export in the presence of the toxin may reflect the
imposition of a rate limit due to folding or maturation of TxVI. Thus, the
prodomain of TxVI can facilitate ER export by a "hitchhiking
mechanism," linking its hydrophobic mature domain to a protein that
traverses the ER efficiently and rapidly. Vps10p family members have been
found across a very broad evolutionary range in multicellular organisms
(20), thus the proposed
mechanism may be evolutionarily ancient and could have been co-opted by
Conus species to allow high expression of hydrophobic conopeptides.
It should be noted that our findings do not rule out prodomain interactions
with additional proteins outside the sortilin family. Screening for
interactors of prodomains from different hydrophobic conotoxins and
structure-function analyses of the TxVI prodomain interaction with Vps10p
family receptors will help define the prevalence of this mechanism in the
secretory pathway. It will also be interesting to see if other eukaryotes
utilize prodomain "tags" to mark secreted proteins with
hydrophobic surfaces as bona fide products that should pass quality
control in the ER.
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FOOTNOTES
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* This work was supported by European Union Grant QLK3-2000-00402 (to M. F.
and A. A.), the Danish Medical Research Foundation and the Carlsberg
Foundation (to C. M. K.), the Germany-Israel Foundation (to Z. E.), and a
grant-in-aid from the Ministry of Education, Science, Sports, Culture and
Technology of Japan (to K. S.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
**
Incumbent of the Daniel Koshland Sr. Career Development Chair at the Weizmann
Institute of Science. To whom correspondence should be addressed. Tel.:
972-8-9344266; Fax: 972-8-9344112; E-mail:
mike.fainzilber{at}weizmann.ac.il.
1 The abbreviations used are: ER, endoplasmic reticulum; TGN, trans-Golgi
network; DMEM, Dulbecco's modified Eagle's medium; Endo-H, endoglycosidase-H;
RRS, Ras rescue system; GFP, green fluorescent protein; MES,
4-morpholineethanesulfonic acid. 
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ACKNOWLEDGMENTS
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We thank Zehava Levy for excellent technical assistance and Dr. Toru Sasaki
for his skilled help with peptide synthesis.
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