The Department of Medical Microbiology and Immunology, University of
Wisconsin-Madison Medical School, Madison, WI 53706, USA
* Present address: Department of Microbiology and Immunology, Stanford
University Medical School, Fairchild Building, D305, Stanford, CA 94305,
USA
Present address: StemCo Biomedical Inc., 2810 Meridian Parkway, Suite 148,
Durham, NC 27713, USA
Author for correspondence (e-mail:
jdbangs{at}facstaff.wisc.edu
)
Accepted 28 May 2002
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Summary |
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Key words: Trypanosome, Lysosome, Flagellar pocket, Endocytosis, LAMP
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Introduction |
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Between the flagellar pocket and the central nucleus reside many membranous
compartments involved in these trafficking pathways, and a growing number of
specific protein markers have been defined. These include a homologue of the
ER molecular chaperone BiP (Bangs et al.,
1993); Rab homologues (Field
et al., 2000
); a clathrin homologue
(Morgan et al., 2001
); and an
unusual glycosylphosphatidylinositol-anchored heterodimeric transferrin
receptor in the flagellar pocket
(Ligtenberg et al., 1994
;
Salmon et al., 1994
). There
are two markers for the terminal lysosomal compartment, trypanopain, the major
soluble thiol protease of trypanosomes
(Caffery et al., 2001
;
Mbawa et al., 1991
), and p67,
a membrane glycoprotein (Brickman and
Balber, 1993
; Kelley et al.,
1999
).
Originally called CB1-gp, p67 was first identified as the ligand for an
N-glycan-specific monoclonal antibody (CB1) generated using total
bloodstream trypanosome ricin-binding proteins as immunogen
(Brickman and Balber, 1993).
p67 has a type I topology (Fig.
1A) with a highly glycosylated N-terminal domain, a 19-residue
transmembrane domain and a 24-residue C-terminal cytoplasmic domain
(Kelley et al., 1999
). It
bears a striking resemblance to mammalian lysosome-associated membrane
proteins (LAMPs), although there are no sequence homologies. In both stages
p67 is synthesized as a 100 kDa N-glycosylated ER species (gp100),
and in bloodstream trypanosomes N-glycan processing in the Golgi
generates the CB1 epitope converting gp100 to gp150. Thereafter it is
delivered to the lysosome, at least in part, by export to the flagellar pocket
followed by endocytosis (Brickman and
Balber, 1994
). In procyclic trypanosomes p67 N-glycans
are not processed and delivery to the lysosome is direct from the Golgi
(Kelley et al., 1995
). Thus
there are several stage-specific aspects to both the processing and the
trafficking of p67.
|
Significant questions remain concerning the structure of p67, its trafficking and turnover, and its subcellular localization. For instance, bloodstream form p67 is cleaved into discrete fragments and degraded upon arrival in the lysosome, but the fate of p67 in procyclic cells is unclear. More interestingly, the signals that mediate stage-specific targeting of p67 remain to be elucidated. By analogy to mammalian LAMPs such signals would be predicted to reside in the cytoplasmic domain. In this work we present a detailed kinetic analysis of the biosynthesis, transport and turnover of p67, including an ordered map of proteolytic cleavages that occur in the lysosome in both stages of the life cycle. In addition, we define p67 localization in relationship to other known markers of the secretory and endocytic pathways. Using this information we then investigate the targeting of p67 by expression of C-terminal truncation mutants in both bloodstream and procyclic trypanosomes. Our results indicate that while lysosomal targeting in procyclic cells occurs in a predictable manner, targeting in bloodstream cells is more complex and may involve novel stage-specific mechanisms.
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Materials and Methods |
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Immunological procedures
Rabbit anti-TbBiP, anti-TbHSP70.4 and anti-VSG antibodies
have been described previously (Bangs et
al., 1996; Bangs et al.,
1993
; McDowell et al.,
1998
). Rabbit antibody to the p67 cytoplasmic domain (anti-CD) was
generated by immunization with a synthetic peptide (KTEEDLLPEEAEGLIDPQN)
coupled to keyhole limpet hemocyanin. Monoclonal anti-p67 (mAb280), which
recognizes and uncharacterized peptide epitope in the p67 lumenal domain, was
a generous gift of David Russell (Cornell University, Ithica, NY). Rabbit
anti-tomato lectin antibody was generated by immunization with purified lectin
(Vector Laboratories, Burlingame CA). Rabbit anti-T. brucei
transferrin receptor antibody was a generous gift of Piet Borst (Netherlands
Cancer Institute, Amsterdam) and rabbit antirhodesain (trypanopain) was a
generous gift of Conor Caffrey (University of California-San Francisco).
Rabbit anti-GFP was a generous gift of Gerard Marriott (University of
Wisonsin-Madison).
Standard immunoprecipitations, surface biotinylation and streptavidin
blotting were carried out as described previously
(Bangs et al., 1997). Gels and
blots were analyzed by phosphorimaging and/or chemiluminescence and final
images were processed in PhotoShop 5.5. For pulse-chase analyses of the
p67
CD and p67
TM reporters extracts of transgenic cell lines were
cycled through three rounds of immunoprecipitation with anti-CD to remove all
endogenous full-length p67 polypeptides (D.L.A. and J.D.B., unpublished). The
remaining p67 reporter polypeptides were then immunoprecipitated with mAb280.
Enzymatic deglycosylation of immunoprecipitates with
peptide:N-glycosidase F was performed according to manufacturer's
instructions (New England Biolabs, Beverly, MA).
Microsequencing
Total membranes of hypotonically lysed MITat 1.4 bloodstream trypanosomes
(2x1010) were extracted in TEN buffer (50 mM TrisHCl, pH 7.5,
150 mM NaCl, 5 mM EDTA) containing detergents (1% NP-40, 0.5% deoxycholate,
0.1% SDS) and protease inhibitors (0.1 mM tosyllysine chloromethyl ketone, 0.1
mM PMSF, 2 µg/ml each of leupeptin, antipain, chymostatin and pepstatin).
p67 polypeptides were immunoselected with an mAb280 IgG column. 600 µg was
fractionated by SDS-PAGE and electrotransfered to PVDF membranes for
microsequencing (Midwest Analytical, St Louis, MO).
Reporter constructs
All reporter constructs were prepared by PCR. Truncated p67 reporters were
generated by placing stop codons before or after the transmembrane domain
(Fig. 1B). Secretory EGFP
reporters (Fig. 1C) were
prepared by in frame fusion of the EP1 procyclin signal sequence
(codons 1-38), EGFP (codons 1-239; Clontech), and the transmembrane and/or
cytoplasmic domains of p67 (codons 617-639 or codons 617-659). All reporter
constructs were cloned into the HindIII/EcoRI sites of the
trypanosomal stable expression vectors, pXS2neo
(Bangs et al., 1996) and
pXS5neo for stable transformation of procyclic and bloodstream
trypanosomes, respectively. pXS5neo is a derivative of pXS2
containing (5'-3', Fig.
1D): 403 bp from the untranscribed 3' end of the rDNA locus;
a 487 bp rRNA promoter element (nts -260 to 227 relative to initiation); a 78
bp splice acceptor fragment (nt -87 to -10 relative to the EP1
procyclin start codon); a multicloning region; a modified 871 bp intergenic
region (nt 22 3' of the aldolase ORF to nt 12 of the downstream orf);
the neomycin phosphotransferase gene; the ß
-tubulin intragenic
region (nts -108 to +532 bp relative to the ß-tubulin stop codon).
Linearized vectors (pXS2, BstXI; pXS5, XhoI) were introduced
into cultured procyclic and bloodstream trypanosomes by electroporation and
stable transformants were selected with neomycin.
Uptake studies
Cultured log phase MITat 1.4 bloodstream trypanosomes were washed,
resuspended (5x107/ml) in serum-free HMI9 medium containing
1% bovine serum albumin, and preincubated (1 hour, 37°C, 5%
CO2). Incubation was continued for 1 hour in the presence of
Alexa488-conjugated transferrin (5 ug/ml, Molecular Probes, Eugene OR) or
biotinyl-tomato lectin (5 µg/ml, Vector Laboratories). For transferrin
uptake P27 (2 µM) was included in both incubations to inhibit degradation
by lysosomal thiol protease. As a control for binding specificity, chitin
hydrosylate (1/1000 final dilution, Vector Laboratories) was included in
duplicate tomato lectin samples. In all experiments cell viability remained
excellent. Following uptake, cells were washed and processed for
immunofluorescence.
Microscopy
Fixed permeabilized procyclic cells were prepared for immunofluorescence as
described previously (Roggy and Bangs,
1999). Cultured bloodstream cells were washed in ice cold PBS with
10 mg/ml glucose, fixed lightly (107 cells/ml, 0.1% formaldehyde in
PBS, 5 minutes, 4°C), centrifuged and resuspended (4x107
cells/ml) in PBS with 5% normal goat serum (NGS). Fixed cells (50 µl) were
smeared on prewashed slides, air dried, extracted sequentially with methanol
and acetone (3 minutes each, -20°C), and then stained as with procyclic
cells. Specific staining was developed with appropriate Alexa488- or
Alexa633-conjugated secondary antibodies, or Alexa488-streptavidin (Molecular
Probes), with 500 ng/ml DAPI. Serial 0.2 µm image Z-stacks were collected
at 100x on a motorized Zeiss Axioplan IIi equipped with a rear-mounted
excitation filter wheel and a triple pass (DAPI/FITC/Texas Red) emission cube.
Fluorescence images were captured with a Zeiss AxioCam B&W CCD camera and
were lightly deconvolved by a nearest neighbor algorithm, psuedocolored, and
merged using OpenLabs 3.0 software (Improvision, Lexington, MA).
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Results |
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To confirm that all p67 glycoforms are encoded in the same open reading
frame, steady state p67 polypeptides were immunoaffinity purified from
bloodstream trypanosomes for N-terminal microsequencing. Peptide sequences
were obtained for gp32 (DATPTVVTVW) and gp42 (SAFVKVVKDD) that unambiguously
indicate cleavages at codons 38 and 241 of the deduced p67 amino acid
sequence. The signal sequence cleavage site (codon 35) of mature gp100 was
previously determined with procyclic p67
(Kelley et al., 1995). In
addition, fragments bearing an intact C-terminus (gp150, gp100, gp75 and gp28)
were identified by reactivity with antipeptide antibody specific for the
cytoplasmic domain (anti-CD; D.L.A. and J.D.B., unpublished). Collectively
these data indicate an ordered map for p67 proteolytic processing
(Fig. 1A) and confirm that all
glycoforms are derived from a full-length p67 precursor polypeptide.
The patterns of p67 fragmentation in both stages of the lifecycle are
strikingly similar given that the full-length precursor glycoforms differ by
50 kDa. To investigate this issue radiolabeled p67 from procyclic and
bloodstream cells were treated with peptide:N-glycosidase F (PNG) to
remove N-glycans. With the exception of gp150, which is absent in
procyclic cells, the sizes of the native glycoforms are closely matched in
both stages of the lifecycle (Fig.
2D, compare lanes 1 and 3). Furthermore, PNG treatment generates a
similar set of deglycosylated polypeptides (dp150 to dp32, compare lanes 2 and
4). As previously demonstrated (Kelley et
al., 1995
), deglycosylation reduces the full-length glycoforms
(gp150 and gp100) to a single 67 kDa species (d150/d100). Assignment of the
deglycosylated gp75 and gp42 species (d75 and d42) is presumed by relative
shifts in electrophoretic mobility. The identical profiles of deglycosylated
polypeptides indicate that cleavage site selection is not stage-specific.
Furthermore, the size similarity of the native fragments in each stage suggest
that the terminal N-glycan modifications that generate the
bloodstream-specific gp150 glycoform are removed rapidly upon arrival in the
lysosome, likely concomitant with proteolytic fragmentation.
Subcellular localization of p67
The steady-state subcellular localization of p67 was investigated by
immunofluorescence with mAb280 to detect p67 and anti-BiP as a marker for the
ER (Fig. 3). In both procyclic
(Fig. 3E) and bloodstream
trypanosomes (Fig. 3F) BiP
localizes to a characteristic network of the ER (green) indicating good
preservation of morphology (Bangs et al.,
1993). In each case, p67 localizes to a prominent vesicular
compartment (red) immediately posterior to the nucleus and well forward of the
flagellar pocket. This pattern is typical, although p67 sometimes presents as
multiple discrete vesicles in the same region
(Fig. 3H). The p67-positive
region also stains for trypanopain indicating that it is a hydrolytic
compartment (Fig. 3G, yellow,
arrowhead). The distribution of p67 relative to total tomato lectin-reactive
polypeptides in bloodstream cells was also assessed
(Fig. 3H). Consistent with
previous work (Nolan et al.,
1999
), tomato lectin detects pNAL-bearing glycoconjugates (green)
throughout the post-nuclear region, which contains many components of the
secretory/endocytic pathways. p67 discretely co-localizes with a subset of the
tomato lectin staining compartments (Fig.
3H, arrowheads).
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Immunoelectron microscopy studies suggest that p67 is a marker for the
terminal endocytic compartment in trypanosomes
(Brickman and Balber, 1993;
Brickman et al., 1995
). We have
confirmed this by immunofluorescent localization of p67 following uptake of
either fluorescent transferrin or biotinyl-tomato lectin as receptor-mediated
cargo in intact bloodstream trypanosomes
(Fig. 4). Endocytosed
transferrin is rapidly degraded in bloodstream trypanosomes
(Grab et al., 1992
;
Steverding et al., 1995
) and
its intracellular detection is dependent on the presence of trypanopain
inhibitors. Even short exposure (2 hours) to such inhibitors results in
significant lysosomal swelling (Fig.
4B,D, compare red inserts). Consequently, endocytosed transferrin
(Fig. 4B, green insert), which
is delivered to the lumen of the lysosome, appears to have a halo of
membrane-bound p67 in the merged image. In contrast, endocytosed tomato lectin
(Fig. 4D) is relatively
resistant to degradation, no protease inhibitor is required, and the patterns
of localization for p67 (red insert) and tomato lectin (green insert) are
completely superimposable in the merged image. The receptor(s) for endocytosis
of tomato lectin is not known, but many endogenous proteins in the flagellar
pocket, including transferrin receptor, have pNAL-containing
N-glycans that could serve as lectin-binding sites
(Nolan et al., 1999
). Whatever
the receptor(s), inclusion of chitin hydrosylate blocks uptake of tomato
lectin (Fig. 4F). These data
clearly demonstrate that endocytosed macromolecules are targeted to, and
degraded in, a compartment for which p67 is a definitive steady state
marker.
|
Generation of truncated p67 reporters
To test our hypothesis that the signals mediating proper lysosomal
targeting reside in the p67 cytoplasmic domain, constructs were engineered
that placed stop codons immediately before or after the transmembrane domain
(Fig. 1B). These reporters
should either be membrane-associated (p67CD) or soluble (p67
TM),
and in both cases would be predicted to be defective in lysosomal targeting if
the relevant signals reside in the cytoplasmic tail. Transgenic procyclic and
bloodstream cell lines stably expressing these reporters, as well as control
cell lines overexpressing wildtype p67 (p67WT), were prepared and analyzed by
three methodologies, pulsechase radiolabeling
(Fig. 5), cell surface
biotinylation (Fig. 6), and
immunofluorescence (Fig. 7). In
the pulse-chase analyses endogenous p67 polypeptides were first removed from
cell lysates with anti-p67 cytoplasmic domain (anti-CD) before specific
immunoprecipitation of the truncation reporters with mAb280.
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p67 targeting in procyclic trypanosomes
Pulse-chase analyses in transgenic procyclic cells confirm the expected
behaviors for the truncation reporters. p67CD is synthesized as a gp100
glycoform that remains cell-associated throughout the 8 hour chase period
(Fig. 6A; 78±6%,
n=4). Little proteolytic fragmentation is apparent and no reporter is
detected in the extracellular medium (D.L.A. and J.D.B., unpublished). Indeed,
p67
CD is very stable in procyclic cells (t
>18
hours; Table 1). The
p67
TM reporter is also synthesized as a gp100 glycoform in procyclic
cells and is largely secreted to the media
(Fig. 5C; 85±6%, 8
hours, n=6) with a rate (t
4.0±0.7 hours;
Table 1) that is within the
range of other soluble reporters in procyclic trypanosomes
(t
1-5 hours) (Bangs et
al., 1996
; Bangs et al.,
1997
). Pulse-chase analyses of procyclic cells overexpressing
wildtype p67 (D.L.A. and J.D.B., unpublished) revealed no significant
alteration in the rate of turnover relative to that of endogenous p67 in
untransformed cells (Table
1).
The localization of the p67 reporters in procyclic cells was investigated
by cell surface biotinylation (Fig.
6A). Cells remained fully viable throughout the biotinylation
procedure and the cytosolic marker Hsp70 was used as a control for cell
integrity. Little or no biotinylated Hsp70 is detected in any procyclic cell
line (Fig. 6A, lanes 2,4,6)
indicating that plasma membrane integrity was maintained throughout the
biotinylation procedure. In untransformed procyclic cells no p67 polypeptides
are detected on the cell surface (Fig.
6A, lane 1). In contrast, cell surface p67 polypeptides are
readily detected in transgenic procyclics expressing either the p67CD
(Fig. 6A, lane 3) or p67WT
(Fig. 6A, lane 5) reporters.
Consistent with the pulse-chase results, biotinyl-p67
CD remains
cell-associated for >24 hours (D.L.A. and J.D.B., unpublished).
These findings were directly confirmed by immunofluorescent localization
(Fig. 7). In addition to the
typical internal staining pattern for endogenous p67 polypeptides, a distinct
halo of cell surface staining is seen in the p67CD cell line
(Fig. 7D) and a faint, but
reproducible, pattern of surface staining is also seen in p67WT cells
(Fig. 7B). No cell surface
staining is ever detected in cells expressing p67
TM or in untransformed
control cells (D.L.A. and J.D.B., unpublished). Collectively these results
argue that the signal(s) for proper targeting of p67 in procyclic trypanosomes
reside in the cytoplasmic domain. Deletion of this domain leads to stable cell
surface localization and additional deletion of the transmembrane domain leads
to quantitative secretion. Furthermore, the existence of a minor pool of cell
surface p67 when the full-length wildtype gene is overexpressed suggests that
the machinery for targeting in procyclic cells can be saturated.
p67 targeting in bloodstream trypanosomes
Striking differences in behavior were found when the various reporters were
analyzed in transgenic bloodstream trypanosomes. As expected, p67CD is
synthesized as a gp100 glycoform and is processed to the corresponding gp150
glycoform (Fig. 5B).
Surprisingly, however, this reporter was then fragmented to the smaller
glycoforms characteristic of delivery to the lysosome. Consistent with this
interpretation, degradation is blocked with the thiol protease inhibitor P27
(D.L.A. and J.D.B., unpublished). Turnover of p67
CD in bloodstream
cells is essentially the same as that of endogenous wildtype p67
(t
0.6±0.1 hour;
Table 1). The p67
TM
reporter is also synthesized as gp100 and processed to gp150, but very little
is actually secreted (Fig. 5D;
5.3±2.3%, at 4 hours, n=6). Most of this reporter is degraded
internally with a turnover rate identical to that of endogenous wildtype p67
(t
0.7±0.2 hours;
Table 1), and P27 blocked
degradation leading to the accumulation of the gp150 glycoform with no
increase in secretion (5.1±2.4% at 4 hours, n=6).
Overexpression of the wildtype p67 gene has no measurable effect on the normal
turnover of total p67 polypeptides (t
0.7±0.1 hours;
Table 1).
Biotinylation assays were performed to detect the presence of these
reporters on the surface of transgenic bloodstream cells
(Fig. 6B). As with procyclic
cells, controls for cell integrity were performed with hsp70
(Fig. 6B, lanes 2,5,8), and
again, no biotinyl-hsp70 is detected. The bloodstream-specific transferrin
receptor was also used as a positive control for biotinylation of proteins
within the extracellular lumen of the flagellar pocket. In all cases a robust
signal is evident for the mature 60 kDa and
42 kDa subunits of this
receptor (Fig. 6B, lanes 3,6,9)
indicating that the relatively sequestered lumen of the flagellar pocket does
not restrict access of the external biotinylation reagent. No biotinylated p67
polypeptides are detected on the surface of either control untransformed cells
(Fig. 6B, lane 1) or on cells
overexpressing wildtype p67 (Fig.
6B, lane 7), but cell surface p67 is consistently present in the
p67
CD cell line (Fig.
6B, lane 4). The presence of a surface pool of p67
CD was
confirmed by immunofluorescence (Fig.
7H). No surface fluorescence is seen on bloodstream cells
overexpressing wildtype p67 (Fig.
7F) or on control and p67
TM expressing cells (D.L.A. and
J.D.B., unpublished). These results differ dramatically from those obtained
with the procyclic cell lines. Low levels of p67
TM and p67
CD do
escape to the medium and cell surface, respectively, indicating that the
cytoplasmic domain contributes to proper targeting in bloodstream
trypanosomes. However, the overwhelming fate of these reporters is degradation
by a P27-inhibitable protease suggesting that lysosomal delivery continues in
the absence of the signal(s) that are required for targeting in procyclic
cells.
Targeting GFP to the lysosome
To determine whether signals in the p67 transmembrane and/or cytoplasmic
domains are sufficient for lysosomal targeting in procyclic cells we fused a
secretory form of green fluorescent protein (GFP) to the transmembrane domain
alone (sGFPCD) or to both domains together
(sGFPWT) (Fig. 1C).
In pulse-chase analyses radiolabeled polypeptides of the expected mass are
detected at equivalent levels in each stable cell line
(Fig. 8, compare lanes 1 and
5). At the end of the chase period recovery of the sGFP
CD
reporter is the same in the absence (Fig.
8, lane 2, 52±6.5%) and presence
(Fig. 8, lane 4,
56±8.6%) of trypanopain inhibitor. Recovery of the sGFPWT
reporter is significantly lower (Fig.
8, lane 6, 24±7.3%, P<0.05), but this loss is
reversed by inhibition of trypanopain (Fig.
8, lane 8, 67±13%, P<0.05). Biotinylation
experiments also reveal both reporters at the cell surface with
sGFP
CD being considerably more abundent than
sGFPWT (K.J.S. and J.D.B., unpublished). The subcellular
localization of the GFP reporters was investigated by immunofluorescence
(Fig. 9). In both cell lines
steady state accumulation of GFP in the ER and nuclear envelope is
consistently observed, as shown by intense colocalization with BiP
(Fig. 9E,G, yellow). The slow
kinetics of GFP folding likely results in ER retention by quality control
machinery (Hammond and Helenius,
1995
; Reid and Flynn,
1997
). In the sGFPWT cell line prominent GFP staining
is also seen in a non-ER post-nuclear region
(Fig. 9G, green, arrowhead),
which is confirmed as the lysosome by robust colocalization of GFP with p67
(Fig. 9H, yellow, arrowhead).
No such colocalization is observed with sGFP
CD
(Fig. 9F, red, arrowhead).
Collectively these results are entirely consistent with the behavior of the
corresponding p67WT and p67
CD reporters and suggest that the p67
cytoplasmic domain targets heterologous reporters to the lysosome in procyclic
trypanosomes.
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Discussion |
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The fate of p67 was originally characterized in bloodstream cells and we
have now extended these studies to the procyclic stage. In bloodstream cells
essentially all ER gp100 is processed to gp150 by N-glycan
modifications in the Golgi. Thus the disappearance of gp100 represents the
rate of transport to the Golgi. However, subsequent transport to the lysosome
must be rapid because p67 fragments can be detected before complete loss of
the gp100 precursor. In procyclic trypanosomes, 100 kDa p67 is transported
without N-glycan modification to the lysosome where it is also
fragmented, probably by trypanopain. The disappearance of gp100 in this stage
represents both the rates of transport to the lysosome and subsequent
proteolysis. Consequently, the measured halftime probably overestimates the
time required for transport. Developmental downregulation of trypanopain
activity in the procyclic stage (Caffery
et al., 2001) probably contributes to the slower rate of p67
turnover, as does the lower growth temperature of procyclics (27°C vs
37°C). Nevertheless, transport of p67 to the lysosome in procyclic cells
is clearly slower than in bloodstream cells, which explains why previously no
degradation of p67 was detected at shorter chase times in procyclics
(Kelley et al., 1995
).
Available evidence suggests that processing of gp100 to gp150, which
generates the CB1 epitope, involves addition of terminal
poly-N-acetyllactosamine (pNAL) to core N-glycans
(Brickman and Balber, 1993;
Nolan et al., 1999
). However,
addition of pNAL cannot fully account for the maturation of gp150
N-glycans. Many proteins of the bloodstream endocytic compartments
have pNAL-containing N-glycans, yet the CB1 monoclonal binds only to
p67 (Brickman and Balber, 1993
;
Kelley et al., 1999
). Thus,
additional p67-specific N-glycan structural feature(s) must
contribute to the CB1 epitope. Interestingly the N-terminal gp32 fragment,
which contains 4 N-glycosylation sites, is not reactive with either
tomato lectin or with CB1 monoclonal (D.L.A. and J.D.B., unpublished). These
findings are consistent with the observation that mature gp150 contains some
N-glycans that are endoglycosidase H-sensitive that is,
unprocessed (Kelley et al.,
1995
).
Despite the glycan-mediated differences in size of mature p67 in bloodstream and procyclic cells, the sizes of the native proteolytic fragments generated upon arrival in the lysosome are remarkably similar. Comparative analyses of the deglycosylated peptides indicate that cleavage site selection is essentially the same in each lifecycle stage. To reconcile these findings we propose the pNAL modifications of p67 N-glycans in bloodstream cells are removed in the lysosome by endogenous glycosidases and that glycan trimming is concurrent with proteolytic cleavage. Consistent with this hypothesis, tomato lectin blots reveal a heterogeneous smear of steady state p67 polypeptides ranging from 150 to 42 kDa (D.L.A. and J.D.B., unpublished). One potential function of the pNAL modifications may actually be to retard p67 turnover in the more robust lysosome of bloodstream cells, and if so, then turnover would be even more rapid without pNAL epitopes.
Previous work using surface biotinylation in pulse-chase experiments
suggested that p67 in bloodstream cells trafficked rapidly through the
flagellar pocket en route to the lysosome. Estimates of peak residence in the
flagellar pocket were as high as 40% of total radiolabeled p67
(Brickman and Balber, 1994),
although later experiments suggest that far less newly synthesized p67 is
present at any given time [see Fig.
9 in Kelley et al. (Kelley et
al., 1995
)]. We have now used surface biotinylation to assess the
steady state level of endogenous p67 polypeptides on the cell surface.
Consistently under conditions where cytosolic hsp70 is not accessible, and
where transferrin receptor in the flagellar pocket is readily detected, no p67
polypeptides are labeled by external biotinylation. Surface biotinylation is
an extremely sensitive assay and this finding indicates that the amount of
total p67 in the flagellar pocket is very low, a finding consistent with
previous antibody-binding studies on intact trypanosomes
(Brickman and Balber, 1993
).
This does not exclude endocytic routing through the flagellar pocket provided
that the rate of transit is rapid; however, it now seems likely that
trafficking directly from the Golgi may be a significant contributing pathway
in bloodstream cells.
p67 targeting signals
The behavior of the p67 truncation reporters in procyclic trypanosomes
indicate that lysosomal targeting motif(s) resides in the 24 amino acid
cytoplasmic domain. Furthermore, the transmembrane and cytoplasmic domains,
but not the transmembrane domain alone, are capable of targeting GFP to the
lysosome indicating that cytoplasmic domain is both necessary and likely
sufficient for correct targeting in procyclic cells. In addition,
overexpressed wildtype p67 leaks to the cell surface suggesting that the
machinery for recognition of the specific lysosomal targeting signals is
saturable. The two dileucine motifs within the cytoplasmic domain are obvious
candidates for such signals (Fig.
1B). In the mammalian endosomal/lysosomal pathway such motifs
mediate targeting of membrane proteins, such as mannose 6-phosphate receptor
(Johnson and Kornfeld, 1992)
and LIMP II (Sandoval et al.,
1994
), by serving as ligands for heterotetrameric adapter protein
(AP) complexes that in turn mediate assembly of clathrin on budding transport
vesicles. Di-leucine motifs can interact with different AP complexes for
basolateral sorting (Heilker et al.,
1996
), for endocytosis
(Heilker et al., 1996
) and for
alternative post-Golgi pathways
(Höning et al., 1998
).
Quantitative and qualitative proof that the p67 di-leucine motifs function in
an analogous manner will require other reporters than those used here and this
work is currently underway. However, homologues of clathrin and ß-adaptin
have been identified in trypanosomes
(Morgan et al., 2001
), and it
is a reasonable speculation that these, along with other adaptin homologues,
will provide the core machinery for p67 targeting.
The behavior of the p67 reporters in bloodstream trypanosomes is more
puzzling. Given the increased endocytic activity of bloodstream cells
(Langreth and Balber, 1975;
Morgan et al., 2001
), and the
fact that p67 can normally traffic through the flagellar pocket, it is not
surprising that overexpressed full length p67 does not leak to the plasma
membrane. However, the efficient lysosomal delivery of the deletion constructs
begs an explanation when they are both quantitatively exported in procyclic
cells. One possibility is that an alternative targeting signal is used in
bloodstream cells. This signal would have to reside in the lumenal domain to
account for lysosomal targeting of p67
TM and could be a
post-translational modification such as the pNAL-containing N-glycans
found in bloodstream cells. Nolan et al. have proposed that pNAL on other
glycoproteins of the bloodstream endocytic pathway, such as the transferrin
receptor, may be a shared epitope specifying internalization via a
hypothetical lectin-like receptor in the flagellar pocket
(Nolan et al., 1999
). Such a
mechanism could mediate endocytic retrieval of the p67 reporters following
delivery to the flagellar pocket. Alternatively, upregulation of a redundant
targeting machinery in bloodstream cells could result in recognition of a
lumenal p67 epitope, peptide or carbohydrate, that is present in both stages.
A second possibility is that lysosomal delivery of the deletion constructs is
a nonspecific consequence of the heightened endocytic activity of bloodstream
cells. In this scenario, p67
CD and p67
TM would enter some
post-Golgi compartment at the intersection of the secretory and endocytic
pathways, perhaps the flagellar pocket or an internal sorting endosome, at
which point they would be carried to the lysosome by `endocytic backflow'.
Whatever the alternative mode of targeting, redundancy would benefit the
parasite by ensuring that no invariant antigen leaks to the cell surface,
where it might elicit potentially lethal host immune responses.
p67 function
p67 has no sequence homology with known mammalian lysosomal proteins, but
its overall structure is analogous to LAMP-1 and LAMP-2, both of which have
type I membrane topologies with multiple pNAL-containing N-glycans
(Hunziker and Geuze, 1996).
LAMPs are thought to form a continuous protective glycocalyx on the lumenal
face of mammalian lysosomes (Granger et
al., 1990
). Surprisingly, gene disruption of mouse LAMP-1
has no effect on basic lysosomal function, although mild effects on brain
tissue are seen in live animals
(Andrejewski et al., 1999
). By
contrast, genetic deficiencies in LAMP-2 correlate with pathological
accumulation of autophagic vacuoles in both humans and mice
(Nishino et al., 2000
;
Tanaka et al., 2000
)
suggesting that it will be possible to assign other functions to individual
lysosomal membrane proteins by genetic strategies. Although the true
function(s) of mammalian LAMPs have not been determined, it may be that p67
plays an analogous role that might also be revealed by genetics. There are too
many gene copies (Kelley et al.,
1999
) for a targeted p67 gene disruption approach to work;
however, gene silencing by inducible expression of double-stranded RNAi
constructs works well in trypanosomes (Ngo
et al., 1998
; Wang et al.,
2000
). Preliminary results with this strategy indicate that
ablation of p67 expression leads to rapid cessation of bloodstream trypanosome
cell growth followed by cell death (D.L.A. and J.D.B., unpublished).
Apparently then, p67 is essential for lysosomal function and in the future
trypanosomes should provide a unique model system for studying the role of
this general class of membrane proteins.
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Acknowledgments |
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