Bloodstream-form
African
trypanosomes
encode
two
structurally
related
glycosylphosph
ati-
dylinositol
(GPI)-anchored
proteins
that
are
critical
virulence
factors,
variant
surface
glyco-
protein
(VSG)
for
antigenic
variation
and
transferrin
receptor
(TfR)
for
iron
acquisition.
Both
are
transcribed
from
the
active
telomeric
expression
site.
VSG
is a GPI
2
homodimer;
TfR
is
a GPI
1
heterodimer
of GPI-anchored
ESAG6
and
ESAG7.
GPI-valence
correlates
with
secretory
progression
and
fate
in bloodstream
trypanosom
es:
VSG
(GPI
2
) is a surface
pro-
tein;
truncated
VSG
(GPI
0
) is degraded
in the
lysosome;
and
native
TfR
(GPI
1
) localizes
in
the
flagellar
pocket.
Tf:Fe
starvation
results
in up-regulation
and
redistribution
of TfR
to the
plasma
membrane
suggesting
a saturable
mechanism
for
flagellar
pocket
retention.
How-
ever,
because
such
surface
TfR
is non-functio
nal
for
ligand
binding
we
proposed
that
it rep-
resents
GPI
2
ESAG6
homodimers
that
are
unable
to bind
transferrin—thereby
mimicking
native
VSG.
We
now
exploit
a novel
RNAi
system
for
simultaneous
lethal
silencing
of all
native
TfR
subunits
and
exclusive
in-situ
expression
of RNAi-resista
nt TfR
variants
with
valences
of GPI
0–2
. Our
results
conform
to the
valence
model:
GPI
0
ESAG7
homodimers
traffick
to the
lysosome
and
GPI
2
ESAG6
homodimers
to the
cell
surface.
However,
when
expressed
alone
ESAG6
is up-regulated
~7-fold,
leaving
the
issue
of saturable
retention
in
the
flagellar
pocket
in question.
Therefore,
we
created
an
RNAi-resista
nt GPI
2
TfR
heterodi-
mer
by
fusing
the
C-terminal
domain
of ESAG6
to ESAG7.
Co-expression
with
ESAG6
gen-
erates
a functional
heterodimeric
GPI
2
TfR
that
restores
Tf uptake
and
cell
viability,
and
localizes
to the
cell
surface,
without
overexpression.
These
results
resolve
the
longstanding
issue
of TfR
trafficking
under
over-expression
and
confirm
GPI
valence
as
a critical
determi-
nant
of intracellular
sorting
in trypanosomes.