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Molecular & Biochemical Parasitology 115 (2001) 1 – 17
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Reviews: Parasite cell Biology: 1
The flagellum and flagellar pocket of trypanosomatids
Scott M. Landfear *, Marina Ignatushchenko
Department of Molecular Microbiology and Immunology, Oregon Health Sciences Uni6ersity, Portland, OR 97201, USA
Received 9 November 2000; received in revised form 26 January 2001; accepted 5 March 2001
Abstract
The flagellum and flagellar pocket are distinctive organelles present among all of the trypanosomatid protozoa. Currently,
recognized functions for these organelles include generation of motility for the flagellum and dedicated secretory and endocytic
activities for the flagellar pocket. The flagellar and flagellar pocket membranes have long been recognized as morphologically
separate domains that are component parts of the plasma membrane that surrounds the entire cell. The structural and functional
specialization of these two membranes has now been underscored by the identification of multiple proteins that are targeted
selectively to each of these domains, and non-membrane proteins have also been identified that are targeted to the internal lumina
of these organelles. Investigations on the functions of these organelle-specific proteins should continue to shed light on the unique
biological activities of the flagellum and flagellar pocket. In addition, work has begun on identifying signals or modifications of
these proteins that direct their targeting to the correct subcellular location. Future endeavors should further refine our knowledge
of targeting signals and begin to dissect the molecular machinery involved in transporting and retaining each polypeptide at its
designated cellular address. © 2001 Elsevier Science B.V. All rights reserved.
Keywords: Trypanosomatid protozoa; Flagellum; Flagellar Pocket; Organelle-specific proteins; Review
1. Introduction
Two distinctive features in the cell biology of trypanosomatid protozoa are the presence of flagella in at
least some life cycle stages and the existence of a
prominent invagination of the plasma membrane called
the flagellar pocket [1]. The purpose of this review is to
provide an overview of recent as well as some longer
standing discoveries concerning the nature of these two
closely apposed organelles. This article is organized
largely around specific proteins that have been shown
to reside in either the flagellum or the flagellar pocket.
The reason for this approach is that a number of
excellent reviews already exist [2 – 6] that deal wholely
or partially with the more general cell biology of the
flagellar pocket and flagellum. In contrast, recently
published material has increased the number of proteins
known to reside within the membranes or lumina of the
flagellum or flagellar pocket and has given us novel
molecular markers with which to probe the biological
functions of these organelles.
The surface membrane of kinetoplastid protozoa,
including Trypanosoma brucei, Trypanosoma cruzi,
Leishmania species, Crithidia fasciculata and others has
been divided into three morphologically distinct subdomains [2]: the flagellar membrane, the flagellar pocket,
and the pellicular plasma membrane (Fig. 1). It is now
recognized that each of these domains represents a
Abbre6iations: BSA, Bovine serum albumen; CRAM, cysteine-rich acidic integral membrane protein; ER, endoplasmic reticulum; ESAG,
expression site associated gene; FCaBP, flagellar calcium binding protein; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol;
HDL, high density lipoprotein; HRP, horse radish peroxidase; IFT, intraflagellar transport; LDL, low density lipoprotein; LPG, lipophosphoglycan; MVT, multivesicular tubule; PFR, paraflagellar rod; PPG, proteophosphoglycan; sAP, soluble acid phosphatase; SDS, sodium dodecylsulfate; Tf, transferrin; TFBP, transferrin binding protein; TLTF, T lymphocyte triggering factor; VSG, variant surface glycoprotein.
* Corresponding author. Tel.: + 1-503-4942426; fax: + 1-503-4946862.
E-mail address: [email protected] (S.M. Landfear).
0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 6 - 6 8 5 1 ( 0 1 ) 0 0 2 6 2 - 6
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Fig. 1. Subcellular structure of a bloodstream form African trypanosome (reproduced from [5] by copyright permission of Elsevier Science Ltd.).
The abbreviations are: az, adhesion zone at the entrance of the flagellar pocket; cv, coated vesicles; er, endoplasmic reticulum; fl, flagellum; fp,
flagellar pocket; gl, glycosome, a membrane bound organelle involved in glycolysis and other metabolic pathways; go, Golgi apparatus; k,
kinetoplast containing highly catenated kinetoplast DNA; l, lysosome; m, mitochondrion; mt, subpellicular microtubules; n, nucleus, sc, surface
coat containing variant surface glycoprotein; tv, tubulovesicular structure. The length of the cell is approximately 20 mm. The cell surface
membrane can be divided into the pellicular plasma membrane surrounding the cell body, the flagellar pocket membrane, and the flagellar
membrane.
highly specialized membrane with distinctive functions
and unique protein and possibly lipid compositions.
Thus, the pellicular plasma membrane surrounds the
body of the cell and is attached to a dense corset of
highly stable, cross-linked microtubules. This membrane contains many of the permeases that mediate
uptake of nutrients via classical transporter cycles, it
provides the cell body with its shape, and in some
cases, it is densely covered with a protein or glycolipid coat that protects the parasite against host immune responses, as in the case of the variant surface
glycoproteins (VSGs) of T. brucei [7] and the abundant glycolipid lipophosphoglycan (LPG) of Leishmania species [8]. The flagellum (Fig. 2) is the classical
motility organelle that moves the parasite forward by
wave-like beats of the microtubule-based flagellar axoneme, but it is also involved in additional biological
activities such as the attachment of parasites to the
endothelium of their insect hosts [1], and it may also
be a specialized sensory organelle. The flagellar
pocket, a deep invagination at the base of the flagellum (Fig. 3) is responsible for uptake of larger nutrients via receptor-mediated endocytosis, for secretion
of proteins into the extracellular medium, and for integration of membrane proteins into the cell surface.
It is noteworthy that these three membranes are physically contiguous, and all constitute part of the
plasma membrane despite their highly differentiated
biological functions. The identification of proteins
that are localized discretely to one of these plasma
membrane components has helped to delineate the
distinct functions of each of these membrane surfaces.
We are now beginning to understand how each compartment is maintained as a unique entity by identify-
Fig. 2. The cytoskeleton of the flagellum and paraflagellar rod of L. mexicana (reproduced from [44], by copyright permission of Elsevier Science
Ltd.). (A) A whole-mount negatively stained cytoskeleton is shown, including the basal body ‘b’ where the flagellum initiates, the flagellar axoneme
‘a’ which is the microtubule-based structure that generates flagellar motility, and the fibrous paraflagellar rod ‘p’ that runs adjacent to the
axoneme. The subpellicular microtubules that lie underneath the pellicular plasma membrane can be seen in the cell body region at the left of the
figure. (B) A transverse view of the flagellar cytoskeleton showing the ‘9+ 2’ arrangement of singlet and doublet axonemal microtubules. The
fibrous paraflagellar rod is underneath the axoneme and consists of the proximal, intermediate, and distal domains (labeled ‘pd’, ‘id’, and ‘dd’,
respectively).
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
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rest of the surface bilayer is the observation that several
well-characterized membrane proteins in various
trypanosomatids are localized discretely in the membrane of this organelle. A number of Ca + 2-binding
proteins of both T. cruzi and T. brucei, receptor-adenylate cyclases of T. brucei, and a glucose
transporter isoform in L. enriettii are all present in the
flagellar membrane, while they occur at low or
undetectable levels on the pellicular plasma membrane.
In addition, earlier biochemical studies [9] detected
specific proteins on SDS-polyacrylamide gels using a
membrane fraction from purified flagella, although this
fraction was not directly compared with the pellicular
plasma membrane to determine which of these proteins
was truly flagellar-specific. Furthermore, studies on the
lipid composition of flagellar membranes of several
micro-organisms [10] have revealed high sterol/
phospholipid ratios compared with other membranes,
and this pattern of differential lipid content appears to
apply in trypanosomatids, as well, where the sterolbinding antibiotic filipin intercalates to a high degree in
the flagellar membrane [9]. A question of central
importance is to determine how proteins are selectively
targeted to or excluded from the flagellar membrane,
and studies on several of the flagellar membrane
proteins are beginning to elucidate this process.
Another intriguing question has to do with the
biological significance of flagellar localization; that is,
what are the specific functions of proteins that reside
exclusively or preferentially in this membrane?
2.1. Flagellar Ca + 2-binding proteins
Fig. 3. A thin section electron micrograph through the flagellar
pocket of a bloodstream form T. brucei parasite showing the contiguous pellicular plasma membrane, flagellar pocket membrane, and
flagellar membrane (reproduced from [6] by copyright permission of
Elsevier Science Ltd.). The flagellum initiates at the basal body near
the base of the flagellar pocket and extends through the anterior
opening of the pocket at the top of the figure. An electron dense
region where the surface membrane apposes the flagellar membrane
constitutes the ‘junctional complex’ or ‘adhesion zone’. The lumen of
the flagellar pocket is filled with diffuse electron dense material. The
solid arrow indicates a cytoplasmic vesicle adjacent to the flagellar
pocket, and the open arrow indicates the kinetoplast DNA that is
enclosed by the mitochondrial membrane and is immediately posterior to the basal body. Subpellicular microtubules can be seen underneath the pellicular plasma membrane at the top and bottom of the
figure, where they have been sectioned at a glancing angle. The scale
bar represents 0.5 mm.
ing targeting signals that sort proteins to each of these
differentiated membranes or to the structures enclosed
by these membranes.
2. Flagellar membrane
The most definitive evidence that the flagellar
membrane is distinct in protein composition from the
Over 10 years ago, Engman et al. [11] identified an
EF-hand flagellar Ca + 2-binding protein, FCaBP, from
T. cruzi. Subsequent studies revealed that FCaBP was
dually modified with myristate on the amino group of
the NH2-terminal glycine and palmitate on the thiol
group of the cysteine at residue 3, and that both of
these acyl groups were required for membrane association and for localization to the flagellum [12]. This
protein appears to be associated with the cytosolic face
of the plasma membrane and does not contain any
apparent transmembrane segments. Furthermore, a fusion protein between the first 24 amino acids of FCaBP
and Green Fluorescent Protein (GFP) was also targeted
to the flagellar membrane, indicating that all of the
essential targeting information was contained within
this NH2-terminal segment of the protein. It is currently
not clear whether the dual acylation that mediates
membrane association is itself responsible for the discrete flagellar localization, via lipid sorting, or whether
a targeting sequence located within the first 24 amino
acids is required for restriction to this organelle. FCaBP
has been shown to associate with the flagellar membrane in a Ca + 2-dependent manner. This property is
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reminiscent of the mammalian protein recoverin [13],
another EF-hand Ca + 2-binding protein from retinal rod
cells that associates with the plasma membrane, when
intracellular Ca + 2 levels rise. Membrane association of
recoverin leads to inhibition of rhodopsin kinase and
thus serves to transmit changes in Ca + 2 levels into a
biological readout [14]. The observations in T. cruzi
reveal a conserved mechanism of regulated membrane
association and suggest by analogy that FCaBP is very
likely to be involved in signal transduction mediated by
changes in intracellular Ca + 2 levels. However, the molecular component of this signaling pathway that is downstream of FCaBP has not yet been identified.
A family of related EF-hand Ca + 2-binding proteins
has also been identified in T. brucei and shown to localize
to the flagellum [15,16]. Hence, these flagellar associated
proteins may be involved in regulating a variety of
cellular processes in various trypanosomatids.
2.2. Receptor-adenylate cyclases
T. brucei [17] and L. dono6ani [18] express a family of
adenylate cyclases that appear to have a large extracellular domain, a single transmembrane domain, and an
intracellular adenylate cyclase domain. These proteins
are likely to serve some function as signal transduction
receptors and are structurally related to the well-characterized mammalian atrial natriuretic peptide receptor
that is a ligand-activated guanylate cyclase [19]. Pays and
colleagues [17] demonstrated by both light and electron
microscopy that a member of this family, ESAG 4, is
localized exclusively to the flagellar membrane, although
what portion of the protein is involved in the organellespecific trafficking is not clear. To date, neither the
ligands for these putative receptors nor the probable roles
of the receptors in signal transduction have been identified.
2.3. Flagellar glucose transporter
Studies on glucose transporters from L. enriettii identified two isoforms, ISO1 and ISO2, that differ exclusively in the NH2-terminal hydrophilic domain that is
thought to be located on the cytoplasmic side of the
surface membrane [20]. Localization of these two isoforms by confocal immunofluorescence and immunoelectron microscopy revealed that ISO1 is restricted to the
flagellar membrane (Fig. 4), whereas ISO2 is located in
the pellicular plasma membrane but not the flagellar
membrane [21]. Furthermore, a chimera between the
unique NH2-terminal domain of ISO1 and another
pellicular plasma membrane glucose transporter, D2,
trafficked to the flagellar membrane, demonstrating that
the ISO1 domain was sufficient for flagellar targeting
[22]. Subsequent deletion and site-directed mutagenesis
identified a sequence of five contiguous amino acids,
RTGTT [23] located between positions 25–29 of the
ISO1 sequence, that was central in flagellar targeting.
Thus, a deletion of 20 amino acids from ISO1 did not
interfere with flagellar targeting, whereas fusing the first
35 amino acids of ISO1 onto ISO2 resulted in flagellar
localization of the chimera, together showing that se-
Fig. 4. Flagellar localization of the glucose transporter ISO1 in L. mexicana. Parasites were transfected with a construct that expresses Enhanced
Green Fluorescent Protein (EGFP) [103] fused onto the COOH-terminus of ISO1. Cells were fixed with methanol and stained with an anti-a-tubulin
antibody followed by anti-IgG antiserum conjugated to Texas Red and examined by fluorescence microscopy. (A) ISO1-EGFP fusion showing
staining (green) on the flagellum and flagellar pocket. (B) a-tubulin staining (red) of the cell body and flagellum. (C) Merged images of (A) and
(B).
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
quence between positions 20– 35 contained a flagellar
targeting sequence. Alanine scanning mutagenesis of
ISO1 showed that the G27A, T28A, and T29A mutants
trafficked less efficiently than wild type transporter to
the flagellar membrane, whereas R25A and T26A
trafficked more efficiently to the flagellum and were
relatively less abundant in the flagellar pocket than the
wild type ISO1. However, addition of the RTGTT
sequence to ISO2 was not by itself sufficient for
flagellar targeting, indicating that the structural context
of this sequence is important for function. These
mutagenesis studies have identified a sequence within
ISO1 that may interact with other components of a
flagellar targeting machinery, and future efforts to
detect potential associations of ISO1 with other
proteins may begin to dissect the targeting pathway.
2.4. GP72, a protein in6ol6ed in flagellar attachment
Null mutants of the gp72 gene of T. cruzi, which
encodes an immunodominant membrane glycoprotein,
revealed a remarkable phenotype: the flagellum was no
longer attached to the cell body after it emerged from
the flagellar pocket, and the motility of the mutants was
reduced [24]. Localization of epitope tagged Gp72
revealed that this protein is distributed over the cell
body surface and the flagellar pocket, but that it is
concentrated in the proximal region of the flagellum
and is undetectable at the distal tip of the flagellum
[25]. Thus, despite its flagellar phenotype, this protein is
quite widely distributed over the parasite surface. It is
possible that Gp72 interacts with the flagellar adhesion
zone, a region where the flagellar membrane adheres to
the pellicular plasma membrane [2], and that the
absence of this interaction could explain the
morphology of gp72 null mutants. Of additional
interest, gp72 null mutants have greatly reduced ability
to establish infections in the insect vector Triatoma
infestans [26].
A homolog of gp72 has been identified in T. brucei
and is designated the fla1 gene [27]. The Fla1 protein is
evenly distributed along the flagellum, except at the
proximal end where the region of the flagellar pocket
stains more strongly. This distribution is similar to that
of a 88 kDa membrane protein from T. brucei that was
earlier observed to concentrate in the flagellar
attachment region [28]. However, unlike Gp72, it was
not possible to directly assess the function of Fla1, as
null mutants could not be obtained and apparently
were not viable.
2.5. Mechanism of targeting to the flagellar membrane
How are proteins like FCaBP, ESAG 4 and ISO1
selectively targeted to the flagellar membrane? In principle, they could either be actively sequestered in this
5
specialized membrane, or they could diffuse into the
flagellum and then be held in the organelle by retention.
Alternatively, the acyl modifications on some proteins
such as FCaBP could cause partitioning into the unique
lipid environment of the flagellar membrane. Furthermore, other proteins like ISO2 are not present in the
flagellar membrane and could be either actively excluded from this compartment or retained over the
pellicular plasma membrane. The answers to these
questions are not yet clear, but some relevant data are
available. Thus ISO1 in which the first 30 amino acids
have been deleted traffics to the pellicular plasma membrane [22], suggesting that this route may be a default
pathway, that operates if no flagellar targeting signal is
present.
Since all surface membrane proteins are thought to
enter the flagellar pocket membrane first [5,6,29], and
since both the flagellar ISO1 and the pellicular plasma
membrane ISO2 are present in the flagellar pocket
membrane, it is likely that this latter subdomain is the
site for differential sorting of flagellar and pellicular
plasma membrane proteins. Presumably, a dominant
flagellar targeting signal causes routing of proteins into
the flagellar compartment, whereas polypeptides that
do not contain this signal may traffic by default to the
membrane surrounding the cell body.
While the machinery responsible for flagellar membrane targeting has not been identified, studies on
flagellar assembly in the green alga Chlamydomonas
reinhardtii may offer some clues about how this process
might occur. An activity designated ‘intraflagellar
transport’ (IFT) [30,31] is responsible for the movement
of newly synthesized components of the flagellar axoneme in both anterograde and retrograde directions.
Large complexes termed ‘rafts’ that contain at least 15
proteins are moved in both directions beneath the
flagellar membrane. Furthermore, the anterograde
movement has been shown to be dependent upon a
kinesin-like protein FLA10, and retrograde movement
is dependent upon a dynein-like protein DHC1b.
Hence, there is a molecular machinery dedicated to
moving complex particles along the flagellum. Although
most of these studies have analyzed movements of
non-membrane proteins, other work has also identified
an apparently related motility process that moves components associated with the flagellar membrane [32,33],
and like IFT, this movement of membrane components
is also inhibited by mutations in the fla10 gene [31,34].
Hence, it is likely that some flagellar membrane
proteins are directed to their correct location by IFT or
a similar process. It is intriguing that three point mutations in the ISO1 flagellar targeting domain resulted in
a phenotype that could be explained by impaired
anterograde transport, whereas two other point mutants had a phenotype reminiscent of impaired retrograde transport [23].
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S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
2.6. Is the flagellum a specialized sensory organelle in
trypanosomatids?
Evidence from other systems representing both the
microbial and mammalian world has highlighted the
involvement of cilia and flagella in sensing the environment. Until recently, this latter function has been less
well appreciated than the better-established role of
these organelles in motility. Thus signal transduction
receptors that mediate cellular responses associated
with mating of C. reinhardtii gametes are located specifically in the flagellar membrane [35]. The complex
family of olfactory receptors in mammals that mediate
the sensation of smell are localized to the ciliary membranes of olfactory neurons [36], and odorant receptors
in Caenorhabditis elegans are also localized to olfactory
cilia [37]. Furthermore, FCaBP and the receptor adenylate cyclases of trypanosomes are very likely involved in
signal transduction and may mediate a flagellar-specific
sensory function. Although the particular function of
the flagellar glucose transporter ISO1 is currently unknown, the recent observations that glucose transporter-like proteins in yeast [38] and other bonafide
transporters in various other micro-organisms and
plants [39–42], as well as the human GLUT1 glucose
transporter [43] can function as signal transduction
receptors to monitor the level of their ligands in the
environment raises the intriguing possibility that this
flagellar isoform might be involved in glucose sensing.
The flagellar localization of various sensory membrane
proteins might be necessary for interaction with downstream components of signaling pathways that could
themselves be localized to the interior of the flagellum.
Further studies on the biological functions of flagellar
membrane proteins should help to elucidate the likely
sensory functions of this organelle.
3. Paraflagellar rod
While trypanosomatids have many conventional
structural components of flagella [4], they also contain
an unusual fibrous body called the paraflagellar rod
(PFR) that is constituted from discrete filaments, runs
along the length of the flagellum, and is attached to the
flagellar axoneme [44] (Fig. 2). In T. brucei, the major
components of the PFR are two closely related proteins
designated PFR-A and PFR-C, each encoded by a
cluster of 4 repeated genes [45]. Similar genes and
proteins have been identified in L. mexicana and T.
cruzi, where they are called PFR-2, PFR-1 and PAR-2,
PAR-3, respectively [4]. This structure, which also occurs in euglenoids, had been speculated to play a role in
motility, but definitive evidence for such a function was
not available until recently, with the development in
trypanosomatids of gene knockout technology using
homologous gene replacement or RNA interference
methods.
A PRF-2 null mutant was generated by homologous
gene replacement in L. mexicana [46]. The mutant
parasites assembled a residual PFR that stained with
antisera against PFR-1, revealing that the PFR is
greatly altered but probably not completely disrupted
in this mutant. Notably, the null mutants had an
approximately 4-fold reduced velocity of forward motility and an altered flagellar beat pattern exhibiting a
reduced wavelength and somewhat lower beat frequency. This motility phenotype might be explained by
reduced elastic bending resistance resulting from alteration of the PFR. In T. brucei, the PFR-A mRNA was
ablated by RNA interference [47] using overexpression
of antisense RNA from an expression construct integrated into the PFR-A gene locus [48]. These functional
null mutants had an even more dramatic phenotype
than the L. mexicana PFR-2 null mutants, as they were
largely paralyzed and sedimented to the bottom of the
tissue culture flask during growth. The PFR appeared
to be disrupted, and PRF-C protein was released into
the detergent soluble fraction, in contrast to its location
in wild type parasites. Together, these two manuscripts
provide the first definitive evidence that the PFR has a
central role in motility of trypanosomatids. A further
understanding of the details of PFR ultrastructure, the
biophysical properties of this rod, and the identity and
function of less abundant PFR proteins [4] should
further clarify the function of this remarkable subcellular structure.
An intriguing study by Gull and colleagues [49] has
identified a segment of the PFR-A protein of T. brucei
that is necessary for targeting to the paraflagellar rod.
Deletion mutagenesis revealed that a region between
amino acids 514 and 570 of this 600 amino acid protein
was essential for addition of PFR-A to the paraflagellar
rod, but this sequence itself was not sufficient to target
GFP to this fibrous structure. Furthermore, the sequence between amino acids 559 and 574 showed substantial homology to similar regions in paraflagellar rod
proteins from other kinetoplastids and from Euglena
gracilis as well as to a sequence in the heavy chain of an
axonemal dynein from C. reinhardtii, implying that a
common signal for flagellar targeting or assembly may
be conserved across these species. More recent studies
revealed that the tripeptide HLA, located at amino
acids 563–565, is shared in common with a novel
actin-related protein, TrypARP, that is also targeted to
the paraflagellar rod and that deletion of this tripeptide
from either PFR-A or TrypARP prevents assembly of
the mutant protein into the rod [50], thus identifying a
probable paraflagellar rod assembly motif. In addition,
PFR-A appeared to have a major site of addition at the
distal tip of the paraflagellar rod but to undergo
turnover along the length of the rod.
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
4. Flagellar pocket
The flagellar pocket is a deep invagination of the
plasma membrane that is located at the base of the
flagellum (Figs. 1 and 3). Although the pocket has a
somewhat distinct morphology in different kinetoplastids [1], its functions are thought to be similar in all
cases. This pocket is located at the anterior end of the
cell, it encloses the base of the flagellum, and it appears
to be surrounded at its opening by a desmosome-like
thickening [3] variously referred to as the ‘junctional
complex’ [2], the ‘zone of adhesion’ [5], or the ‘maculae
adherens’ [6] (Fig. 3). It has been proposed but not
demonstrated that this junctional complex may restrict
the flow of material into and out of the flagellar pocket,
but macromolecules can clearly move both into and out
of the pocket (see below).
It has been appreciated for some time that the flagellar pocket membrane, representing between 0.4 and 3%
of the cell surface [2,6], is highly specialized and is the
only known site for endocytosis, for secretion of
proteins from the cell, and for addition of integral
membrane proteins to the cell surface [5,6,29]. Since
these vesicle-dependent events are restricted to a very
small component of the surface membrane yet bloodstream trypanosomes can internalize a membrane area
equivalent to that of the flagellar pocket approximately
every 2 min [51], the flagellar pocket is probably the
most active organelle known for endocytosis [5]. While
a dense network of subpellicular microtubules is attached to the cytoplasmic side of the pellicular plasma
membrane [4] (Figs. 2 and 3), these microtubules are
absent from the flagellar pocket, with the exception of
a quartet of specialized microtubules that run along one
surface of the flagellar pocket [6]. It is thought that the
subpellicular microtubule network may prevent processes such as vesicle fusion and receptor-mediated
endocytosis from occurring at any place outside the
flagellar pocket, but the molecular mechanisms that
restrict these events to the flagellar pocket are not
known and could well be more complex than simple
physical exclusion by the cytoskeletal network. In contrast, invaginations resembling the coated pits of mammalian cells have been observed by various groups [5]
in the membrane of the flagellar pocket. In addition,
studies on uptake of several ligands have revealed that
horseradish peroxidase (HRP), ferritin, and BSA-gold
are taken up by non-saturable fluid-phase endocytosis
that is relatively slow [51], whereas transferrin (Tf) and
low density lipoproteins (LDL) are endocytosed by a
rapid, saturable, and ligand-specific process similar to
receptor-mediated endocytosis in mammalian cells.
Both types of endocytic process appear to be restricted
to the flagellar pocket membrane. Furthermore, although only examined for a few cases, both membrane
bound [29] and secreted proteins [52] appear to reach
7
the cell surface at the flagellar pocket membrane, underscoring the role of this membrane in delivery of
proteins to the cell surface and exterior. Finally, the
lumen of the flagellar pocket can be seen to contain
electron dense material (Fig. 3) and clearly harbors
specific proteins [52] that are secreted into this space.
All of these observations support the conclusion that
the flagellar pocket is a highly differentiated component
of the plasma membrane that is specialized for uptake
and secretion of molecules required by or released from
the parasite.
Although an exhaustive review of secretion and endocytosis in trypanosomatids is beyond the scope of the
current article, Sections 4.1and 4.2 below provide an
overview of these processes as they relate to the flagellar pocket. Subsequent sections review specific proteins
that are known to be components of the membrane or
the lumen of the flagellar pocket. The identification and
functional investigation of such organelle-specific markers will likely provide us with a deeper understanding of
the structure, assembly, dynamic activity, and function
of this unusual subcellular structure that is one of the
hallmarks of the Kinetoplastida.
4.1. Secretory pathway
Neither the secretory nor endocytic pathways are
nearly as well understood in trypanosomatid protozoa
as they are in mammalian cells or yeast, but the current
level of knowledge suggests that there are many broad
parallels. In particular, there is an extensive endoplasmic reticulum (ER) that is widely dispersed throughout
the cytoplasm [29,53], there is a Golgi apparatus consisting of 4– 6 flattened stacks, there is a ‘budding zone’
of vesicles between the ER and the cis-face of the
Golgi, there are flattened cisternae, tubulovesicular elements and coated vesicles adjacent to the Golgi that
probably constitute the trans-Golgi network, and there
are various larger vesicles ( 100 nm) between the
trans-Golgi and the flagellar pocket that are thought to
be transport vesicles [54] (Fig. 5). Trafficking of the
abundant variant surface glycoproteins (VSGs) has
been studied in bloodstream African trypanosomes [29],
where these glycosylphosphatidylinositol (GPI) anchored proteins form a dense coat that covers the
surface of the parasite and protects it from the host
immune system [55]. Immunogold labeling of thin
cryosections demonstrated the presence of VSG in the
ER, all of the Golgi cisternae, the trans-Golgi network,
and the transport vesicles, suggesting that newly synthesized VSG traffics sequentially through these organelles
on its way to the flagellar pocket and cell surface, which
are also heavily labeled.
Studies on the extracellular enzyme secreted acid
phosphatase (sAP) in promastigotes and intracellular
amastigotes of L. dono6ani, the major secreted protein
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Fig. 5. Ultrastructure of L. mexicana promastigotes prepared by high-pressure freeze substitution and Epon embedding showing a variety of
organelles involved in secretion or endocytosis (reproduced from [54] by copyright permission of The Company of Biologists Ltd.). (A) Anterior
end of the cell showing the Golgi (g), the ER (er), the electron dense budding zone (bz) with vesicles on the cis-side of the Golgi and with
translucent vesicles (v) on the trans-side of the Golgi. (B) ER-derived vesicles in the budding zone (bz) appear to form new Golgi cisterna. (C)
Vesicles filled with particles (asterisks) near the trans-side of the Golgi. Four coated vesicle-like structures (arrowheads) budding from the most
trans-side cisterna of the Golgi. (D) Large spherical translucent vesicle (v) adjacent to the flagellar pocket. Below is an enlarged detail of the vesicle
membrane (large arrowhead) displaying a regularly arranged coat-like structure (small arrowheads). Tubules are often connected to large
translucent vesicles (v, arrow and also arrows in E and F). (E and F) Clusters of regularly arranged tubules (t) located close to the Golgi (g) and
the flagellar pocket (fp) showing tubules in longitudinal section (E) and cross section (F). (G) Coated pit-like invaginations of the flagellar pocket
membrane. bz, budding zone; er, endoplasmic reticulum; fl, flagellum; fp, flagellar pocket; g, Golgi; k, kinetoplast; n, nucleus; t, clustered tubules;
v, translucent vesicles. Bars are 0.5 mm (A –F) and 100 nm (inset D and G).
of this parasite, have underscored the role of the Golgi
in modification of secreted proteins. Immunofluorescence localization revealed that sAP was present dif-
fusely in the cytoplasm and in a more concentrated
form in the flagellar pocket, and the enzyme was ultimately secreted into the medium [56], suggesting that it
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
is transported via a constitutive secretory pathway to
the lumen of the flagellar pocket and thence to the
extracellular space. The extracellular enzyme was
present as two heterodisperse bands of 110 and 130
kDa on SDS-polyacrylamide gels but was synthesized
as two intracellular precursors of discrete sizes, 92.5
and 107 kDa [57]. Addition of monensin, a classical
inhibitor of Golgi function, caused morphological
changes to the Golgi apparatus, as earlier shown in T.
brucei [29], and suppressed the heterodisperse mobility
of the sAP bands [58], apparently by inhibiting a Golgispecific carbohydrate modification. This and other [52]
work has demonstrated that complex phosphoglycans
are added to sAP in the Golgi complex and is responsible for the diffuse mobility on SDS-polyacrylamide
gels, and that similar modifications occur on other
secreted macromolecules including the abundant glycolipid lipophosphoglycan (LPG) and on a complex of
proteoglycans designated proteophosphoglycans (PPG)
[59]. Furthermore, a GDP-mannose transporter required for addition of mannose to complex carbohydrates such as those on LPG is localized to the Golgi,
where it imports from the cytoplasm into the Golgi
lumen the nucleotide sugar precursor used in synthesis
of the oligosaccharide chains [60]. Hence as in mammalian cells, the Golgi is responsible for complex carbohydrate modifications during biosynthesis of secreted
and membrane bound proteins and other macromolecules.
Little is known about the precise nature of vesicles
that mediate transport of secreted and membrane
proteins between the ER, the Golgi, and the flagellar
pocket. In general few markers are available for such
vesicles, and in vitro fusion systems have not been
developed to allow dissection of components required
for vesicle docking and fusion. However, genes encoding rab protein homologues, small G proteins involved
in regulating vesicle fusion in various steps of the
exocytic and endocytic pathways of mammalian cells
[61], have been cloned from T. brucei [62] supporting
the likely conservation of vesicle fusion mechanisms
between these protozoa and higher eukaryotes.
4.2. Endocytic pathway
All endocytic events, either receptor-mediated or
pinocytotic, appear to initiate at the membrane of the
flagellar pocket. Invaginations resembling coated pits of
higher eukaryotes can bee seen pinching off from the
flagellar pocket surface [51,54] (Fig. 5G) and are likely
to be the precursors for endocytic vesicles. Following
exposure of bloodstream trypanosomes to antisera
against VSG, to HRP, or to labeled Tf, BSA, or
ferritin, a variety of internal vesicles and tubulovesicular networks were labeled with the internalized components [51,63–68]. These membrane bounded bodies
9
ranged in diameter from 20 to 200 nm, were located
between the flagellar pocket and the nucleus, and are
believed to represent the early and late endocytic compartments. In addition, internalized labeled material
also appears in electron-dense vesicles thought to be
lysosomes. Coated vesicles from T. brucei, at least some
of which are likely to be endocytic, have been isolated
and partially characterized. These vesicles contain a
minor protein component with the same mobility on
SDS-polyacrylamide gels as bovine brain clathrin, but
this protein does not cross-react with antibodies directed against mammalian clathrin, and its identity is
uncertain [69]. An apparent integral membrane protein
of 77 kDa has been isolated from these vesicles, and
antisera against the gel-purified protein labels vesicles in
thin sections of trypanosomes, suggesting that this
polypeptide is a marker for endocytic vesicles [70].
A number of studies have also been performed on
endocytosis in promastigotes and amastigotes of several
Leishmania species. Overath and colleagues [54] have
applied an improved sample preparation method of
high pressure freezing and freeze-substitution to visualize vesicles and tubules in the anterior region of L.
mexicana promastigotes. In addition to observing improved images of organelles involved in secretion, such
as the ER, the Golgi, intermediary transit vesicles, and
large translucent vesicles close to the flagellar pocket
(Fig. 5), they were able to identify various membranous
structures that labeled with the fluid phase marker
HRP, with biotinylated surface markers, or with antisera against several membrane proteins or oligosaccharides and thus represent the endocytic pathway. In
addition to a rich array of vesicles and tubules similar
to those observed earlier, a thicker tubule 100– 200
nm in diameter was observed that extended the length
of the cell, was itself filled with smaller vesicles, and was
labeled with internalized components. This multivesicular tubule (MVT) labeled with antisera directed against
both a transmembrane form and a GPI-anchored form
of acid phosphatase and with the GPI-anchored surface
protease GP63, but it did not label with an antibody
directed against the protein component of sAP or the
secreted PPG. These latter results suggest that MVT is
not part of the secretory pathway, as had been proposed originally (at the time of the initial discovery of
the MVT) on the basis of light microscopic data relating to overexpression of dolichol–phosphate–mannose
synthase, a normally ER-resident enzyme involved in
GPI biosynthesis [71]. Rather the MVT appears to be
part of the endocytic machinery that internalizes surface proteins that are expressed at relatively high levels,
including the overexpressed proteins used in these studies [54,71,72]. Presumably, proteins transit from the
flagellar pocket to the MVT via other vesicles and
tubules observed by Overath and colleagues. Similar
conclusions have been reached by Dwyer and col-
10
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
leagues [72] by studying high level expression in L.
dono6ani promastigotes of GFP fusion proteins containing either the transmembrane domain of 3% nucleotidase/nuclease or the GPI addition signal of GP63.
These tagged fusion proteins trafficked to the cell surface but were also found in an MVT compartment
running the length of the cell. Fluid phase and particulate markers such as dextrans and positively charged
nanogold particles, used to track endocytosis in higher
eukaryotes, were also internalized into the same MVT
strongly suggesting an endocytic nature for this
compartment.
As in higher cells, lysosome-like organelles appear to
be the end stage of the endocytic pathway for many
internalized components. In the L. mexicana family,
two lysosomal enzymes, cysteine proteinase and arylsulfatase [73], are localized to large electron dense organelles originally designated ‘megasomes’ [74] that are
considered to be the unusual lysosomes of these parasites. Furthermore, biotinylated markers such as dextran or b-glucorinadase were internalized by L.
mexicana infected macrophages and subsequently entered the parasitophorous vacuole and the flagellar
pocket and were thence targeted within the parasite to
the megasomes [75].
4.3. LDL receptors and the CRAM protein
LDL is required by African trypanosomes for robust
growth and apparently is the major source of cholesterol for these parasites [76]. Early biochemical studies
[51] demonstrated that LDL uptake is saturable, can be
competed by unlabeled LDL, is temperature- and
Ca + 2-dependent, and occurs at a very rapid rate compared with fluid-phase uptake. Furthermore, gold labeled LDL adhered to the flagellar pocket membrane
and was also present in intracellular vacuoles, suggesting that a specific LDL receptor was located in the
flagellar pocket and mediated internalization of this
lipoprotein. A T. brucei protein of 86 kDa, apparently
a fragment of a larger 145 kDa protein [77], has been
purified by LDL affinity chromatography and proposed
to be the parasite LDL receptor [76], although it has
also been suggested [78] that this polypeptide may be a
contaminant that co-purifies with the true receptor. A
polyclonal antiserum against the purified fraction
stained the flagellar pocket, and much of the stain
appeared to be in the lumen of the pocket.
In separate work [79], the gene for the cysteine-rich,
acidic, integral membrane protein (CRAM) was cloned
Fig. 6. Localization of CRAM protein in the flagellar pocket (fp) membrane of a procyclic (insect form) T. brucei cell (reproduced from [79] by
copyright permission of the American Society for Microbiology). The electron dense spots adjacent to the membrane are 15 nm gold particles that
were used for indirect immunolabeling of CRAM protein. The arrow marks the flagellum.
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
and shown to encode a protein with multiple 12 amino
acid repeats that have substantial sequence similarity to
the repeat units in the NH2-terminal region of the human
LDL receptor. Furthermore, CRAM is located on the
flagellar pocket membrane (Fig. 6), as determined by
immunoelectron microscopy, and the repeat units appear
to be on the lumenal side of this membrane. Hydropathy
analysis of the sequence revealed the presence of a single
putative transmembrane segment downstream of the
repeat units followed by a hydrophilic COOH-terminal
domain of 41 amino acids. CRAM RNA is expressed at
about a 5-fold higher level in procyclic trypanosomes
compared with bloodstream forms, and the CRAM
protein is also expressed more abundantly in procyclic
parasites. On the basis of the similarity to mammalian
LDL receptors, it has been suggested [80] that CRAM
might be a parasite receptor for a lipoprotein, possibly
high density lipoprotein (HDL).
The higher level of expression of CRAM in procyclic
parasites compared with bloodstream forms has raised
an interesting question. Coated pits and vesicles typically
associated with receptor-mediated endocytosis have only
been observed in bloodstream and not procyclic trypanosomes [64], and it has been suggested that receptormediated endocytosis may be of minimal importance in
the insect stage of the life cycle, where CRAM is
expressed at the higher level. However, studies on procyclic trypanosomes have demonstrated that antiCRAM IgG can bind to and be endocytosed by the
CRAM protein during this life cycle stage [80]. These
studies reveal that endocytosis followed by degradation
of the ligand does occur in procyclics, possibly via
vesicles of distinct morphology from those seen in the
mammalian stage of the life cycle.
An important study on CRAM by Lee and colleagues
[81] has contributed to our understanding of targeting
and retention of flagellar pocket membrane-specific
proteins. These authors observed that a deletion of either
eight or 19 amino acids from the COOH-terminus of
CRAM caused the protein to be retained in the ER,
where it largely localized with the ER marker Bip.
Unexpectedly, deletions of either 29 or 40 amino acids
from the COOH-terminus partially restored trafficking
past the ER but allowed the surface protein to reach the
flagellum and pellicular plasma membrane, as well as the
flagellar pocket membrane. The authors suggest that a
signal for transit through the ER is present in the first
eight amino acids and that a signal for retention in the
flagellar pocket is encompassed by the first 29 amino
acids. Although the precise explanation for the behavior
of the deletion mutants is not clear, removal of both
signals apparently allows some of the protein to traffic
to the cell surface but not to be retained at the flagellar
pocket. Consistent with the presence of targeting information within the 41 amino acid COOH-terminal domain, a fusion between the TrpE protein and this
11
cytoplasmic domain of CRAM traffics to the flagellar
pocket. Intriguing problems that remain to be solved are
to define the precise amino acids that constitute the
flagellar pocket retention signal and to identify the
proteins that presumably interact with this signal to
prevent further migration of CRAM over the surface of
the parasite.
4.4. The T. brucei transferrin receptor
Early biochemical studies demonstrated that Tf is
taken up by a mechanism resembling receptor-mediated
endocytosis and that Tf-gold was localized to the flagellar
pocket with staining largely in the lumen of this organelle
[51]. Subsequent work by Schell et al. [82] succeeded in
purifying a 42 kDa Tf-binding protein (TFBP) by Tfaffinity chromatography of solubilized membranes from
T. brucei bloodstream forms that was ultimately identified as the product of the expression site-associated gene
7 (ESAG 7) [83] that is located in telomeric expression
sites upstream from expressed copies of the VSG genes
[7]. The open reading frame of ESAG 7 is closely related
to that of ESAG 6, and subsequent studies [84] confirmed
that the functional TFBP is a heterodimer of ESAG 6
and ESAG 7. Thus expression of both gene products was
required for Tf binding when expression studies were
performed in Xenopus oocytes [85], insect cells [86], or
procyclic trypanosomes [87]. ESAG 7 is not an integral
membrane protein, and ESAG 6 is modified by a GPI
moiety. The receptor oligomer is apparently held into the
membrane by the ESAG 6 GPI anchor. Furthermore,
immunoelectron microscopy studies [85,87,88] with antisera directed against ESAG 6 or ESAG 7 revealed
staining largely in the lumen of the flagellar pocket but
also on the flagellar pocket membrane. Further support
for TFBP as a bonafide Tf receptor comes from the
observation that TFBP complexed to Tf is routed to the
lysosomes where the Tf is degraded but the TFBP is
recycled [84].
The striking difference between the structure of the
oligomeric GPI-linked T. brucei TFBP and the mammalian Tf receptor, which is a transmembrane protein,
suggests that these two types of receptor function in
fundamentally different ways. First, classical receptors
involved in endocytic events contain a cytoplasmic domain required for ligand-dependent internalization [89],
whereas neither ESAG 6 nor ESAG 7 possesses such a
domain. It is possible that the TFBP interacts with
another transmembrane protein to transmit the ligandinduced signal for internalization. Furthermore, the
consistent observation that TFBP is largely present in the
lumen rather than in the membrane of the flagellar
pocket is puzzling and raises the question of how such
a protein might function. Possibly, TFBP is released into
the lumen to bind Tf and then either re-enters the
membrane or transfers its ligand to membrane bound
12
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
Fig. 7. Fibrous material in the lumen of the flagellar pocket detected in a deep-etch, freeze fracture image of the flagellar pocket of an L. mexicana
promastigote (reproduced from [52] by copyright permission of The Rockefeller University Press). The filaments (arrowheads) are composed of
particles with a similar size and periodicity to those of purified sAP and thus may represent in situ polymers of this protein. The arrow points
toward the opening of the flagellar pocket, and fl marks the flagellum.
receptors. Thus fundamentally important questions that
remain to be answered are — how is the TFBP internalized, how is it retained in the flagellar pocket, and
what is the relationship between apparently soluble and
membrane bound TFBP? The mechanism of Tf uptake
is another example of the intriguing differences between
these single cell parasites and their more complex mammalian hosts.
4.5. Possible hemoglobin receptor
Leishmania parasites require hemin for growth, but
they are able to grow on blood agar medium without
addition of hemin, suggesting that they can acquire this
nutrient from hemoglobin. Biochemical and ultrastructural studies [90] have demonstrated that promastigotes
of L. dono6ani bind 125I-hemoglobin with high affinity,
that this binding can be specifically competed with
unlabeled hemoglobin, and that the hemoglobin binds
initially to the flagellar pocket membrane and is subsequently internalized in vesicles and degraded. Affinity
chromatography led to the isolation of a 46 kDa
protein derived from a membrane fraction that bound
to hemoglobin agarose but whose binding could be
specifically competed with unlabeled hemoglobin.
Hence, this 46 kDa protein may be a hemoglobin
receptor that is localized to the flagellar pocket
membrane.
4.6. Proteins secreted into the lumen of the flagellar
pocket
Since proteins destined for secretion reach the cell
surface at the flagellar pocket, the lumen of the pocket
functions as an intermediate transit zone between the
cytoplasm and the extracellular space, and work from
various groups has demonstrated that several secreted
proteins accumulate in the pocket before their release.
Immunofluorescence studies on the abundantly released
sAP of L. dono6ani promastigotes revealed that this
enzyme accumulates to a high concentration in the
flagellar pocket and that release from the pocket was
dependent upon energy and may be coupled to flagellar
beating [56]. A conserved sAP is secreted by many
species of Leishmania [91] and by both promastigotes
and amastigotes [92]. Overath and colleagues [52] have
also studied sAP extensively in L. mexicana promastigotes. In this species, sAP consists of a 100 kDa glycoprotein associated non-covalently with a high molecular
weight proteoglycan, and assembles into striking long
curved filaments that can be imaged by electron microscopy and are composed of bead-like subunits.
These sAP filaments can be found abundantly in the
lumen of the flagellar pocket (Fig. 7) and are secreted
into the extracellular medium. The authors suggest that
monomers or oligomers are initially secreted into the
pocket where they assemble into the filaments and
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
ultimately exit the pocket. One hypothesis emerging from
these studies is that the flagellar pocket lumen is a site
for assembly of polymeric macromolecular structures,
much the way that collagen fibrils and other components
of the extracellular matrix assemble in extracellular
spaces in mammalian tissues. In contrast, the sAP of L.
dono6ani promastigotes was non-polymeric and revealed
globular mono- or oligomers in negatively stained electron micrographs [93]. Cloning of sAP genes from L.
mexicana identified two genes, lmsap1 encoding the
major 100 kDa sAP and lmsap2 encoding the minor 200
kDa sAP [94]. In both enzymes, Ser/Thr-rich domains
were the sites for extensive phosphoglycan modification.
Similarly two tandemly repeated sAP genes, SAcP-1 and
SAcP-2, were cloned and sequenced from L. dono6ani
and shown to encode isoforms with regions of high
sequence identity, including Ser/Thr-rich domains believed to be the sites of phosphoglycan addition [95].
A second type of filamentous structure originally
called the ‘network’ is clearly distinct from sAP, and is
also secreted into the flagellar pocket lumen [52]. Network material emerging from the mouth of the flagellar
pocket appears as a meshwork in the center of clusters
of promastigotes that adhere to each other in culture and
in the insect and may be responsible for formation of
these large cellular aggregates. These network fibers stain
with a monoclonal antibody directed against a carbohydrate epitope that is also present in sAP, but they do not
stain with a monoclonal antibody directed against the
polypeptide component of sAP. More recently, the
network filaments of L. major have been studied at the
molecular level. Purification of material secreted into the
medium followed by structural analysis revealed a very
unusual proteophosphoglycan (PPG) that ran as a diffuse, high molecular weight species on SDS-polyacrylamide gels, contained large amounts of mannose,
galactose, arabinose, and phosphate, and whose protein
component consisted of 87 mol% of glycosylated phosphoserine, serine, alanine, and proline [96]. The glycans
isolated from PPG were also components of the major
surface glycolipid LPG but were organized differently,
and they were also present in sAP, indicating that both
glycoproteins undergo the novel Golgi-mediated glycosylation. In vitro this purified material formed long (up to
6 mm), cable-like, unbranched filaments that were indistinguishable from the ‘network’ filaments earlier identified. In L. major there are multiple genes encoding
related PPGs, and one of these, ppg1, was the first to be
cloned and sequenced [97]. The predicted protein product
of this gene ( 2300 amino acids) contains NH2-terminal
and COOH-terminal domains with conventional amino
acid sequences separated by a region containing 100
repetitive peptides consisting exclusively of alanine, serine, and proline that are the sites of phosphoglycan
attachment. Remarkably, the ppg1 gene product is a
membrane bound protein (mPPG) that is modified by a
13
GPI anchor and is distributed over the pellicular plasma
membrane, indicating that secreted PPG must be encoded by other members of this gene family. Subsequent
work [59] has led to the cloning of candidate sequences
for the secreted filamentous PPG, now designated fPPG,
and a nonfilamentous PPG secreted by amastigotes,
designated aPPG.
Elegant in vivo studies [98] have established a likely
role for secreted fPPG in the parasite-vector interaction.
Both L. mexicana infecting Lutzomyia longipalpis and L.
major infecting Phlebotomus papatasi form parasite aggregates in the thoracic midgut and stomodeal valve that
are held in place by a gel-like plug. Immunoelectron
microscopy revealed that this plug consists of filamentous networks that entangle and immobilize the promastigotes and that stain with antisera that are specific
for PPG. This matrix is proposed to both retain the
parasites within the insect gut following digestion of the
blood meal and to block access between the foregut and
the midgut during a subsequent blood meal. The consequent difficulties the sandfly experiences engorging the
blood lead to multiple probing of the host accompanied
by increased disgorging of infectious metacyclic forms
that are largely free of the network and located anterior
to it. Hence, the promastigotes secrete filamentous material from the flagellar pocket that profoundly influences
the vector-parasite interaction to promote parasite transmission.
Finally, a recent study [99] has demonstrated that both
GPI-linked proteins that are otherwise anchored into the
plasma membrane and phospholipase C-cleaved GPIlinked proteins that have had their lipid anchors removed
are present in the lumen of the flagellar pocket. Antisera
specific for the cleaved GPI anchor (earlier called crossreacting determinant) react with flagellar pocket components both before in vitro treatment with phospholipase
C (representing species whose anchors were earlier
cleaved in vivo) and after in vitro treatment with this
enzyme (representing intact GPI-containing species that
were cleaved in situ within thin sections). It is not clear
how GPI-containing proteins, including the Tf receptor
and VSG, are released from the membrane into the lumen
of the pocket or what function they might serve there,
but they may exist as micellar structures explaining their
solubility in an aqueous environment.
4.7. Trypanosome T lymphocyte triggering factor
In studies on the interaction of T. brucei parasites with
their mammalian hosts, a parasite gene was isolated [100]
that encodes a protein designated T lymphocyte triggering factor (TLTF) that induces CD8+ T cells to secrete
interferon-g. This protein does not contain an apparent
signal sequence and hence does not appear to follow the
classical biosynthetic pathway for a secreted, vesicular,
or integral membrane protein. Fluorescence microscopy
14
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
localization using GFP fusions to TLTF revealed that
this protein is targeted to the region of the flagellar
pocket at the base of the flagellum. More detailed
localization by immunoelectron microscopy showed that
the fusion protein was enclosed in electron dense bodies
that subtended the flagellar pocket [101]. Thus although
TLTF appears not to be a membrane protein, it is present
inside ovoid shaped bodies that may be vesicular and that
are closely associated with the flagellar pocket membrane. Furthermore, these electron dense bodies cluster
at the anterior side of the flagellar pocket, suggesting that
TLTF interacts asymmetrically with the flagellar pocket
and also implying that the flagellar pocket membrane is
likely to be non-uniform in structure. The biological
significance of this subcellular location is not clear, nor
has the function of TLTF been determined.
Donelson and colleagues have investigated the region
of TLTF responsible for the flagellar pocket-associated
targeting by preparing chimeras between segments of
TLTF and GFP, and they have identified a large segment
spanning amino acids 114– 257 of this 453 amino acid
protein that is responsible for the described localization.
Random mutagenesis of this targeting domain revealed
many mutations that did not affect targeting and other
mutations scattered throughout the domain that did
affect localization. Thus, the 144 amino acid domain
appears to represent a three dimensional (3-D) structural
targeting signal and clearly does not encompass a short
contiguous motif of the type responsible for localization
of many organelle-specific proteins. This region of TLTF
is predicted to have a high a-helical content and a high
probability of forming a coiled-coil, suggesting that
secondary structure of this type could be involved in the
routing of this polypeptide. One very intriguing result is
that a subset of the targeting mutations cause mislocalization of the mutant protein not to the cytoplasm but
to the flagellum, with especially strong staining at the
anterior tip of this organelle. The authors suggest the
possibility that TLTF normally interacts with subpellicular microtubules during its trafficking to the flagellar
pocket, and that some disruption of a retention signal in
the aforementioned mutants allows them to continue to
traffic along the adjacent flagellar microtubules, resulting
in their movement along the body of the flagellum to its
anterior tip.
More recent investigations [102] have shown that
TLTF is a cytoskeleton-associated protein that fractionates quantitatively with the insoluble cytoskeletal pellet
upon lysis of cells with nonionic detergent. Furthermore,
the protein appears to be associated with the flagellar
fraction of the cytoskeleton, as solubilization of the
pellicular microtubules with Ca + 2 does not extract
TLTF but leaves it behind in a pellet containing the
flagellar axoneme, paraflagellar rod, and other associated
fibrous structures such as the flagellar attachment zone
and a quartet of specialized microtubules that line the
anterior face of the flagellar pocket. Hence, TLTF
appears to target to the anterior cytosolic face of the
flagellar pocket by specific association with a subset of
cytoskeletal components. The results with TLTF highlight the complexity of subcellular targeting events that
exist in trypanosomatids and broaden the scope of
current investigations to include a protein that probably
does not associate directly with a membrane.
5. Overview and future directions
The last few years have seen a mini-explosion in the
identification of proteins that target to the flagellum and
flagellar pocket in trypanosomatid protozoa. We now
have available several reasonably well-characterized
proteins that are routed to various components of each
organelle, and in some cases, we have information on the
sequence within the protein that is responsible for the
subcellular localization. There are a number of issues
raised by these studies that are now ripe to be addressed.
In the first instance, it will be important to identify the
cellular machinery that interacts with each protein and
that delivers it and/or retains it at the correct intracellular
site. However, one potential difficulty of genetic strategies will be to design screens or selections that will detect
mutations that alter delivery of flagellar or flagellar
pocket proteins. Targeting processes that are operative
in other organisms, such as IFT, can be investigated in
trypanosomatids to determine whether they are responsible for routing of proteins to the flagellum or flagellar
pocket in these protozoa as well. It will also be interesting
to determine how widely targeting signals can be recognized among different trypanosomatids or even in other
organisms. The unique structure of these protozoa suggests that there may be some highly specialized systems
that have been elaborated by these parasites to construct
and maintain their own distinctive organelles. However,
it is also likely that some of the routing pathways brought
to light in these ancient eukaryotes will prove to be
relevant to distantly related organisms, as was brilliantly
proven with the initial discovery of GPI anchors in
trypanosomes [55] and their subsequent identification in
mammalian cells.
Another area of active investigation is to further
elucidate the biological functions of flagellar and flagellar
pocket proteins. In most cases, we have hints or partial
knowledge about possible activities but not a clear
picture of the specific biochemical or physiological functions or the reasons for the sequestration of the proteins
within each organelle. Thus, the ISO1 protein can
operate as a glucose transporter, but we do not yet know
why it is targeted to the flagellar membrane, while
another very similar permease traffics to the pellicular
plasma membrane. While the flagellar receptor-adenylate
cyclases and the flagellar calcium-binding proteins are
S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17
almost certainly involved in signal transduction, we do
not know what initial signals they respond to nor the
particular pathways in which they are involved. It has
now been determined that the flagellar rod proteins are
operative in some significant way in flagellar motility, but
how they contribute to this activity is not at all clear.
While CRAM is most likely involved in receptor-mediated endocytosis, the ligand recognized by this putative
receptor has yet to be definitively identified. The role of
TLTF in the biology of the trypanosome is still a mystery,
as it is unlikely to serve the parasite well by simply
activating T cells to secrete g-interferon. Nonetheless, it
is likely that further research on these well-defined
‘markers’ of flagella and flagellar pockets will give us
considerable additional insight into the specialized functions of each organelle. Thus, we have been alerted to the
probable function of flagella in sensing the extracellular
environment in addition to their ‘conventional’ function
in parasite motility. We are likely to gain a deeper
appreciation for the multiple activities of these complex
organelles that must often serve biological functions in
these ‘simple’ unicellular eukaryotes that can be achieved
by multiple differentiated cells in metazoan organisms.
Acknowledgements
This work was supported by grant number AI25920
from the National Institutes of Health. S.M.L. is a
Burroughs Wellcome Molecular Parasitology Scholar,
and M.I. is a postdoctoral fellow of the American Heart
Association, Northwest Chapter.
[10]
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