 |
INTRODUCTION |
Sorting of newly translated lysosomal proteases may occur by two
different mechanisms. In mammalian cells, the predominant is through
the addition of phosphomannosyl residues and targeting to the lysosome
pathway by binding to M6P1
receptors within the Golgi (1). However, transport of lysosomal enzymes
in many cells is unaffected by a deficiency in the phosphotransferase, which is required for M6P synthesis (2). M6P-receptor-independent membrane association has been reported for several lysosomal proteins (3). Confirmation and further analysis of a M6P-independent sorting
pathway in mammalian cells has been complicated by both the presence of
the M6P pathway itself and difficulty in distinguishing effects on
protein folding versus protein trafficking when deletional mutants of the protease precursors were analyzed (4, 5). Trypanosoma cruzi and Leishmania mexicana are
protozoa (Trypanosoma) representing one of the earliest
lineages of eukaryotic cells. They nevertheless express proteases
homologous to cathepsins L and B and, unlike yeast, have an organelle
ultrastructure more reminiscent of mammalian cells (6). While the
cathepsin L-like protease prodomains of these organisms
have significant homology to mammalian procathepsin L (e.g.
45% identity for cruzain versus mouse cathepsin L), there
are no carbohydrate modifications, so they are unique experimental
models for analyzing M6P-independent protein sorting.
Trypanosome cysteine proteases are synthesized as precursor proteins
with a hydrophobic signal peptide, a 100-122-amino acid prodomain, a
200-210-amino acid catalytic domain, and, in most cases, a
100-130-amino acid carboxyl-terminal domain (7-12). The function of
the carboxyl-terminal domain is as yet unknown. Hypotheses that it
plays a role in protease inactivation, or in facilitating folding of
the catalytic domain, have been ruled out by expression of fully active
recombinant proteases without this domain (10). Other proposed
functions have included mediating intracellular trafficking of the
protease (13), immune evasion (14), and facilitating activity against
specific macromolecular substrates (15).
The prodomain of cysteine proteases has two well defined functions,
maintaining the enzyme in an inactive form (zymogen) until it reaches
an appropriate site of protease function, and functioning as a
structural template to ensure proper folding during translation (10).
The prodomain can also act as a reversible inhibitor and a stabilizer
of the mature protease (16) and has also been implicated in protease
precursor trafficking (17, 18). A hypothesis for the role of the
prodomain in trafficking suggests that a peptide motif near the amino
terminus is recognized and bound by a membrane receptor within the
Golgi (18). This receptor-prodomain interaction is proposed to direct
protease precursors to appropriate cellular compartments. Release of
free, active catalytic domain from the prodomain-receptor complex is
thought to occur in a downstream compartment and require a final step
of proteolytic processing.
We analyzed the contribution to intracellular protease trafficking of
each of the three major domains of the T. cruzi cathepsin L-like protease, "cruzain," by transfection of
constructs containing coding regions for the domains coupled to GFP as
a reporter gene. We show that the prodomain is necessary and sufficient
for trafficking. A nine-amino acid sequence motif, homologous to the
putative membrane binding region of procathepsin L (18), was identified
and its function confirmed by site-directed mutagenesis. Finally, the requirement of proteolytic release of the catalytic domain from the
prodomain-receptor complex was confirmed by analysis of mutants in
which the prodomain/catalytic domain processing site was altered. The
results of these studies suggest that a protein motif trafficking pathway for lysosomal proteases arose early in eukaryotic cell evolution and may continue to function in higher eukaryotes independent of the M6P pathway.
 |
EXPERIMENTAL PROCEDURES |
Culture Conditions--
L. mexicana (MNYC/BZ/62/M379)
promastigotes were grown at 27 °C in RPMI 1640 (Life Technologies,
Inc.) containing 10% FBS (Sigma) and 10% liver infusion tryptose
medium, and T. cruzi epimastigotes (MHOM/BR/78/Sylvio-X10-CL7) were cultured in liver infusion
tryptose/FBS at 27 °C (33). Mid-log cultures containing from 1 to
5 × 107 cells were used in all experiments.
Oligonucleotides--
The synthetic DNA oligonucleotides used
were synthesized on a Perkin-Elmer Applied Biosystems 394 DNA
synthesizer at the Biomolecular Resource Center (University of
California San Francisco) (see Table
I).
PCR Amplification Reactions--
PCR amplifications were for 25 cycles in a volume of 50 µl, containing 2 units of Pwo
polymerase, 0.1 µg of each primer, and 2 ng of plasmid DNA as
template on a Perkin-Elmer DNA thermal cycler. Amplification conditions
were: 1-min denaturation at 94 °C, 1-min annealing at 55 °C,
2-min extension at 72 °C.
Construction of pT-GFP and pT-Pre-GFP--
The green fluorescent
protein coding sequence was generated by PCR amplification using
pS65T-C1 (CLONTECH) as DNA template and GFP1 and
GFP2 oligonucleotides as primers. For pT-Pre-GFP we used GFP3 instead
of GFP1. GFP3 contains the signal peptide of cruzain. The amplification
product containing a multicloning site at the 3' region of the GFP gene
was treated with SpeI and ClaI restriction
enzymes, gel-purified, and then ligated into the appropriate site in
the pTEX plasmid (26) to generate pT-GFP or pT-Pre-GFP.
Construction of pT-Pro-GFP and pT-ProCat-GFP (see Diagrams in
Figs. 1 and 3)--
The prodomain (Pro) and prodomain-catalytic domain
(ProCat) of the cruzain gene were amplified from pCheYTc (10) using the oligonucleotide primers Pro1 and Pro2, and Pro1 and Cat2, respectively. A SpeI site was incorporated in all the primers. Following
digestion with SpeI, the PCR products were gel-purified and
ligated into pT-GFP. This procedure resulted in the generation of Pro
and ProCat coding regions of cruzain fused upstream of the GFP coding
sequence in the pT-GFP vector. The fused domains were in-frame with
the GFP sequence, as verified by DNA sequencing. The specific sequences from cruzain in each case were
Leu115 to
Thr14 for pT-Pro-GFP and
Leu115 to
Gln195 for pT-ProCat-GFP. Both of these constructs included
the cleavage site between the pro- and the catalytic domains, but
neither included the cleavage site between the catalytic domain and the
carboxyl-terminal domains. Amino acids are numbered based on the papain
numbering system (34).
Construction of pT-GFP-Ctd--
The carboxyl-terminal domain
(Ctd) of the cruzain gene (Tyr186 to Leu342)
contained in pCheYTc (10) was PCR-amplified using oligonucleotide primers Ctd1 and Ctd2. Either HindIII or BamHI
sites were included in the primers to facilitate cloning of the
resultant PCR product into the multicloning site of the GFP gene in
pT-Pre-GFP. This amplification product was digested and ligated as
described above.
Construction of pT-Pro'Cat-GFP--
A PCR-based mutagenesis
procedure was used to generate V3D and V2P substitutions at the
cleavage site of the prodomain (Fig. 3D). Two separate PCR
reactions were done with primers Pro1 and Mut2 in one reaction and
primers Mut1 and Cat2 in a second reaction (27). These reactions were
carried out using 1 ng of plasmid pCheYTc and the PCR conditions
described above. Two DNA fragments were generated by the reactions.
Following agarose gel electrophoresis, these fragments were recovered
and pooled, and 1 µl of the mixture was used in a second round of PCR
amplification with primers Pro1 and Cat2 and an annealing temperature
of 60 °C. A resulting 1-kilobase product was cloned into pT-GFP to
generate pT-Pro'Cat-GFP.
Transfection--
The transfection procedure used was a
modification of that described by Hariharan et al. (28).
Parasite cultures were grown to mid log in the appropriate media
supplemented with 10% FBS. Cells were harvested by centrifugation and
washed twice in electroporation buffer (100 mM NaCl, 3 mM KCl, 5 mM Na2HPO4, 2 mM KH2PO4, 0.5 mM
MgCl2, 0.1 mM CaCl2) and
resuspended at 108 cells/ml in electroporation buffer.
Four-hundred fifty microliters of cell suspension was incubated on ice
for 10 min in a cuvette containing 10 µg of plasmid DNA in 50 µl of
water. The cells were electroporated with a single pulse using a
Transfector-600 (BTX Corp., San Diego, CA) set at 400 V and 500 microfarads using an electrode with a 0.8-mm gap. The electroporated
cells were left on ice for a further 10 min and then diluted into a 5 ml of liver infusion tryptose/FBS. The parasites were incubated for
48 h before adding 20 µg/ml G418 (Geneticin, Life Technologies,
Inc.). Transformants were selected by gradually increasing the G418
concentration to either 100 µg/ml for L. mexicana or 200 µg/ml for T. cruzi over 3 weeks. Parasites were
subcultured every 5 days in the presence of G418 and analyzed as populations.
Detection of GFP/Confocal Microscopy--
Cell pellets were
washed twice with phosphate-buffered saline (4 °C, 10 min, 3000 rpm)
and fixed in 0.1 M sodium cacodylate buffer, pH
7.4, containing 2% paraformaldehyde and 1% sucrose. GFP fluorescence
was first confirmed in fixed cells with a Zeiss Axioplan fluorescence
microscope (excitation and emission of fluorescein). Each trypanosome
preparation was imaged with a laser scanning microscope 410 (Carl Zeiss
Inc., Thornwood, NY) equipped with an Axiovert 100 microscope (Zeiss),
a 63
, 1.4 NA plan-APOCHROMAT objective lens (Zeiss), and an
argon/krypton laser. The fluorescein-labeled probe and the propidium
iodide were imaged separately. Alternatively, samples were imaged with
an MRC 1024 unit (Bio-Rad) equipped with a Nikon Diaphot 200 inverted
microscope. Propidium iodide was imaged using the 568-nm laser line and
collecting emissions longer than 590 nm. For all specimens, a series of
two-dimensional slice images, 0.5 µm apart were acquired starting
above the top surface of the section and extending below the bottom
surface. A zoom factor of 2.0 was used during the scanning, resulting
in a voxel size of approximately 0.2 µm for each specimen. Each slice
voxel intensity was the average of at least three successive scans. Images were transferred to a UNIX workstation for archiving and analysis.
Digital image cytometry was done using the Quantitative Image
Processing System in the Laboratory for Cell Analysis at the University
of California San Francisco Cancer Center (29). The Quantitative Image
Processing System images were acquired using a fluorescent Zeiss
Axioscope equipped with a Xillix 1024 CCD camera and an automated
filter wheel to select emission wavelengths. All mechanical controls
for the system were under software control on a SUN workstation. Images
were acquired sequentially for each fluorochrome, in addition to a
bright field image, to show morphological features.
Western Blot--
Western blots were performed with 1:1000
dilutions of the anti-GFP monoclonal antibody
(CLONTECH) as the primary antibody, in
phosphate-buffered saline, 5% non-fat milk, and detected using horseradish peroxidase-conjugated secondary antibody and ECL Western blot analysis system (Amersham Pharmacia Biotech).
Immunoelectron Microscopy--
Immunoelectron microscopy was
performed on frozen sections from epimastigotes fixed in 2%
paraformaldehyde and 0.1% glutaraldehyde in phosphate-buffered saline,
pH 7.4. Sections were probed with either rabbit antibody raised against
recombinant cruzain (10, 21), conjugated to 15-nm gold (Amersham
Pharmacia Biotech), or mouse anti-GFP monoclonal antibody
(CLONTECH), conjugated to 10-nm gold. Controls were
conducted with the nontransfected cell line and by omission of the
respective primary antibody.
Lysosomal Localization--
Epimastigotes were stained for 30 min with 20 nM red LysoTracker (Molecular Probes,
Eugene, OR), a fluorescent probe (577 nm absorption, 590 nm emission)
used to investigate protein localization in the lysosomes because of
its selective accumulation in cellular compartments with low internal
pH. Fixed and LysoTracker-stained epimastigotes were observed in a
Zeiss microscope equipped with UV epifluorescence.
Homology-based Model of Cruzain Prodomain--
The coordinates
of human procathepsin L (Protein Data Bank deposition code 1CJL (22)
were used to model the proregion of cruzain. Coordinates were
visualized with the program CHAIN (30). Optimal rotomer conformation
for side chains of the cruzain sequence was selected based on the
orientation of the corresponding side chain in the procathepsin L
structure. These choices corresponded to a statistically likely rotomer
conformation in all instances (31). Fig. 4 was generated with MOLSCRIPT
(32).
Mutagenesis at the Conserved Nine-amino Acid Prodomain
Motif--
All substitution mutants were constructed using a PCR-based
method (27) as above. The annealing temperature was varied empirically to maximize the yield of the products. The primers used to replace specific amino acids in each mutant are given in Table
II.
The extreme primers (Tex1 and Cat2) were common for the secondary
reactions in all the mutants. The amplified fragments were gel-purified, digested with SpeI, and ligated in place of
the corresponding wild type fragment in the pT-ProCat-GFP vector.
 |
RESULTS |
Expression of GFP in L. mexicana Promastigotes and T. cruzi
Epimastigotes--
GFP was expressed in both trypanosomes using the pT
vector system (Fig. 1A).
Leishmania promastigotes expressing GFP required 2 weeks of
selection in G418, while T. cruzi epimastigotes required 3 weeks. Expression of GFP alone (Fig. 1, A and B),
without associated protease domains, resulted in distribution of the
fluorescent protein throughout the cytoplasm of promastigotes or
epimastigotes, including the flagellum, as observed previously in
transfection of the related parasite, Leishmania major
(19).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Confocal microscopy of T. cruzi
(A, C, E, and
G) and L. mexicana
(B, D, F, and
H). Each image is the average of at least three
successive 0.5-µm optical slices. Red color is propidium
iodide (E-H) to identify nucleus and kinetoplast, which are
just anterior to the lysosome/endosome compartment (see Ref. 21). The
following plasmid constructs were used: pT-GFP (A and
B), pT-GFP-Ctd (C and D), pT-Pro-GFP
(E and F), pT-ProCat-GFP (G and
H). Below the confocal images is a schematic representation
of the protein domains included in the plasmids. Fl,
flagellum; K, kinetoplast; L, lysosome/endosome
compartment; N, nucleus. Bar = 10 µm.
|
|
The Carboxyl-terminal Domain of Cruzain Does Not Direct GFP to L/E
Compartments--
Fig. 1, C and D, show that
constructs, which included the carboxyl-terminal domain, the last 20 amino acids of the catalytic domain, the natural processing site
(VVGGP) between the catalytic domain and carboxyl terminus (downstream
of GFP), and the signal peptide (
Leu115 to
Ala105) (upstream of GFP), did not alter the diffuse
cytoplasmic localization of the fluorescent protein.
The Prodomain Is Sufficient for Directing GFP to L/E
Compartment--
Fig. 1, E and F, show that GFP
is correctly directed to the L/E compartment of both T. cruzi and Leishmania when it is expressed downstream of
the cruzain prodomain and ProCat processing site (
Leu115
to Thr14). Identical trafficking is observed if the entire
cruzain catalytic domain is also included (Fig. 1, G and
H). Intense fluorescence is seen in the "megasome"
compartment of Leishmania promastigotes and the
"reservasomes" of T. cruzi epimastigotes. These are both late endosome- or lysosome-like vacuoles identified by their size, ultrastructure, pH, and position within the cell relative to propidium iodide-labeled nucleus and kinetoplast (20, 21). Localization to these
compartments was confirmed by colocalization with a fluorescent marker
of low pH cellular compartments, red LysoTracker (Fig. 2, A and B), and by
im- munoelectron microscopy with both an antibody to GFP and an
antibody to the catalytic domain of cruzain (Fig. 2C).

View larger version (98K):
[in this window]
[in a new window]
|
Fig. 2.
Confirmation of localization of ProCat-GFP in
lysosome/endosome compartment (A) of T. cruzi by confocal microscopy with simultaneous staining with
red LysoTracker (B). T. cruzi
epimastigotes were transfected with pT-ProCat-GFP as described under
"Experimental Procedures." Bar = 10 µm.
C, confirmation of colocalization of native protease and GFP
fusion protein (ProCat-GFP) in lysosome-like organelles
(arrows) of T. cruzi by immunoelectron microscopy
with both an antibody to GFP (10-nm gold particle) and an antibody to
cruzain (15-nm gold particle). T. cruzi epimastigotes were
transfected with pT-ProCat-GFP as described under "Experimental
Procedures." Bar = 1 µm.
|
|
The Prodomain Must Be Removed for the Final Steps of Protease
Sorting--
While a prodomain-membrane receptor interaction is
proposed for proper trafficking of mammalian procathepsin L from
endoplasmic reticulum or the Golgi compartment to lysosomes (3), the
catalytic domain must eventually be cleaved from the prodomain to
release soluble active protease (3). The effects of specific cysteine protease inhibitors on the ultrastructure of T. cruzi in
culture suggested that the detrimental effects of the inhibitors are
due to inhibition of autoproteolytic cleavage of the prodomain from the
catalytic domain in a late Golgi compartment (21). In the presence of
cysteine protease inhibitors, unprocessed cruzain accumulated in the
Golgi and did not reach the L/E compartment. To test the hypothesis
that the prodomain must be removed prior to final sorting of cruzain to
this compartment, a mutant construct (Fig.
3D) was analyzed in which the
processing site between prodomain and catalytic domain was altered.
Western blot of T. cruzi extracts (Fig. 3E)
showed that the protein product of the transfected construct was not
processed to the catalytic domain-GFP fusion protein seen in wild type
organisms. Fig. 3, A
C, show that failure to cleave the prodomain
resulted in abnormal accumulation of GFP in the Golgi compartment at
the expense of normal L/E targeting.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 3.
Immunolocalization (A and
C) and confocal microscopic image (B)
of GFP fusion protein in T. cruzi epimastigotes
transfected with pT-Pro'Cat-GFP (mutated processing site).
Immunoelectron microscopy was performed using an antibody to GFP (10-nm
gold particle). Note diminished localization to lysosome/endosome
(L) and accumulation in Golgi (G). C
is a higher magnification to highlight accumulation of cruzain/GFP in
Golgi stacks. The shift of cruzain/GFP to Golgi (G) from
lysosome (L) is shown in B by confocal
microscopy. K, kinetoplast; N, nucleus.
Bar = 1 µm (A and C) or 10 µm
(B). Diagram and sequence of mutant is shown in
D. E, Western blot showing incomplete processing
of Pro'Cat-GFP mutant. Extracts from 1 × 107
stationary epimastigotes were used for SDS-polyacrylamide gel
electrophoresis and Western blot using anti-GFP monoclonal antibody.
Note accumulation of unprocessed precursors in second lane
(mutant). Molecular mass is shown in kilodaltons on the
left.
|
|
Alignment of Trypanosome and Mammalian Cysteine Protease Prodomain
Sequences Identifies Putative Membrane Receptor Binding
Motifs--
The M6P-independent sorting pathway for mammalian
procathepsin L is thought to involve specific motifs within the
prodomain interacting with a microsomal membrane receptor (3).
Alignment of the kinetoplast prodomains with that of mammalian
procathepsin L (Fig. 4A)
reveals similarity in a nine-residue sequence, which, in procathepsin
L, mediates prodomain-membrane association (3). A homology-based model
of the prodomain, based on the crystal structure of procathepsin L
(22), shows this motif is on a solvent-exposed loop between helices 1 and 2 at the amino terminus (Fig. 4B).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Top, alignment of prodomains showing
homology between mammalian and trypanosome cysteine proteases. Note
nine-amino acid motif (41-50) implicated in prodomain-membrane
interaction (box) (18). The alignment was generated using
the PileUp algorithm of the Genetics Computer Group package (version
8.1) with a gap weight of 12 and a gap length weight of 4. The
following GenBankTM and SwissProt sequences were aligned:
P06797 (mouse cathepsin L), P07711 (human cathepsin L), M84343
(cruzain), and Z14061 (L. mexicana). Bottom:
model of prodomain of cruzain built based on homology to structure of
procathepsin L (22) and statistically likely rotomer conformation (see
"Experimental Procedures"). Note position of conserved motif on
solvent-exposed loop (green, Lys44 to
Ala52) near the amino terminus of the proenzyme. Residues
numbered are those chosen for mutation (Fig. 5).
|
|
Mutagenesis of the Nine-amino Acid Motif Confirms Its Importance to
Lysosomal Protease Trafficking--
Based on the assumption that the
arginine- and histidine-rich receptor binding motif would likely form
salt bridges with negatively charged residues on the receptor, and
because glutamic acid replacements in the motif would be most
disruptive, five mutations were constructed and evaluated. All changed
neutral or positively charged amino acids to glutamic acid. Two mutated
residues were in helices flanking the loop region containing the target
motif (K42E at the end of helix 1 and A53E at the beginning of helix 2, Fig. 4). Fig. 5 shows that, despite
changing these amino acids to glutamic acid, normal targeting of GFP to
the L/E compartment was observed. Three mutations within the loop
(K44E, G46E, and R47E) all resulted in distribution of GFP throughout
the cytoplasm of the cell (Fig. 5), similar to the distribution of GFP
when GFP is expressed alone or with the COOH-terminal domain (Fig. 2).
No retention in the endoplasmic reticulum was observed with any of the
constructs, as would be expected if protein misfolding occurred.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 5.
Mutations within prodomain amino-terminal
loop, but not in flanking helices, alter
trafficking of protease precursor. Glutamic acid substitutions
were chosen to produce maximal disruption of salt bridges between the
positively charged prodomain loop and presumed negatively charged
receptor motifs. Note normal localization of GFP with mutations in
residues flanking the loop, but mis-sorting with mutations within loop.
Example of confocal images for K42E and R47E. The right
panel in each pair of photos is phase contrast to show outline of
organisms.
|
|
 |
DISCUSSION |
We have shown that the prodomain of the cathepsin
L-like cysteine protease, cruzain, is necessary and
sufficient for directing GFP to the L/E compartment of two primitive
eukaryotic unicellular organisms, T. cruzi and L. mexicana. In constructs lacking any protease domain, GFP was
distributed throughout the cytoplasm, including the flagellum. Addition
of the carboxyl-terminal domain failed to alter this distribution,
indicating that the carboxyl-terminal domain is not sufficient to
direct proper protein sorting. Correct sorting with the prodomain
construct occurred whether or not the catalytic domain was included.
Coupled with previous analysis of Leishmania cysteine
protease gene products (20, 23), these results suggest that neither the
carboxyl-terminal domain nor the catalytic domain is required in
trafficking of trypanosome cysteine proteases, at least in the
extracellular stages of T. cruzi and L. mexicana.
The pT-cruzain/GFP constructs correctly directed GFP in both L. mexicana and T. cruzi. This result suggests that key
aspects of the cysteine protease trafficking pathway are shared between the two organisms. In fact, alignment of the kinetoplastid prodomains showed both significant homology between the prodomains of the two
parasite species, as well as significant similarity to mammalian cathepsin L in a nine amino acid sequence implicated in mouse procathepsin L binding to microsomal membranes (3). Site-directed mutagenesis experiments confirmed that this motif, located on a solvent
exposed loop near the amino terminus of procathepsin L (22), must be
intact if proper protease sorting is to take place.
Mammalian procathepsin L binds to a microsomal membrane receptor via
its prodomain, which is later cleaved to release soluble mature
protease into specific cellular compartments (18). Previous work by
Eakin et al. (10) indicated that the prodomain of cruzain is
autoproteolytically removed from the catalytic domain at pH 5-6 at the
processing site indicated in Fig. 3 (10, 24). To test whether this
autoproteolysis step was necessary for final intracellular sorting, we
prepared constructs in which the normal prodomain/catalytic domain
processing site was mutated to prevent proteolytic cleavage by cruzain.
By Western blot, the transfected gene product was not processed (Fig.
3) and the prodomain remained attached to the catalytic domain.
Expression of this mutated protease-GFP construct resulted in a
population of T. cruzi epimastigotes in which GFP now
accumulated within the Golgi compartment. Based on these results, as
well as previous studies showing that synthetic cruzain inhibitors
cause accumulation of precursor cruzain in the Golgi (21), we propose
that removal of the prodomain occurs in an acidified late Golgi or
early post-Golgi compartment. A similar location for proteolytic
processing has been observed for the lysosomal glycosidase of another
primitive eukaryote, Dictyostelium discoideum (25).
The protozoa T. cruzi and L. mexicana are closely
related species in one of the earliest lineages of eukaryotic cells. As experimental models they can provide a glimpse of basic protein sorting
pathways that might have developed early in the evolution of the
eukaryotic cell. By analogy to a model of M6P-independent trafficking
of the lysosomal proteases cathepsin D and cathepsin L in mammalian
cells (3, 17), we propose that a membrane-associated receptor binds the
prodomain of cruzain after its exit from the endoplasmic reticulum,
presumably early in its Golgi transit. Evaluation of this model in
primitive eukaryotic cells like T. cruzi is more
straightforward because the cruzain prodomain lacks not only M6P, but
any carbohydrate moiety (13). The observation that the nonglycosylated
prodomain of the T. cruzi cysteine protease is necessary and
sufficient for directing GFP to the L/E compartment of either organism
is consistent with the model proposed by McIntyre and Erickson (17) for
trafficking of mammalian procathepsin L. There is significant sequence
similarity among these proteases in the nine-amino acid motif proposed
to mediate procathepsin L binding to microsomal membranes (18). The
observation that this motif is present on a solvent-accessible loop
(22) and the results of the mutagenesis experiments reported here
support the hypothesis of its role in prodomain-receptor interactions and the presence of a conserved M6P-independent pathway of lysosomal protease trafficking.