Department of Molecular Sciences, University of Tennessee Health Science
Center, 858 Madison Avenue, Memphis, Tennessee 38163, USA
* Present address: Department of Medicine, Washington University School of
Medicine at Barnes-Jewish Hospital, St Louis, MO 63110, USA
Author for correspondence (e-mail:
jcox{at}utmem.edu)
Accepted 6 November 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sorting signal, Actin, Recycling, TGN, Late endosomes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the precise sorting pathway followed by newly synthesized chicken
AE1 anion exchangers has not been defined, previous studies have shown that
the variant chicken kidney AE1-4 anion exchanger has the capacity to undergo
recycling to the Golgi following delivery to the plasma membrane. By contrast,
the AE1-3 variant, which lacks the N-terminal 63 amino acids of AE1-4, does
not recycle to the Golgi (Adair-Kirk et
al., 1999). Mutagenesis studies have further shown that a
tyrosine-dependent sorting signal within the N-terminal 63 amino acids of
AE1-4 is necessary both for Golgi recycling and for efficient basolateral
sorting of this variant anion exchanger in transfected MDCK cells
(Adair-Kirk et al., 1999
). The
tyrosines at amino acids 44 and 47 of AE1-4 were critical for these sorting
activities and for the association of AE1-4 with elements of the actin
cytoskeleton. Tyrosine 47 of AE1-4 resides within the sequence YVEL, which is
conserved among all characterized AE1 anion exchangers except for chicken
AE1-3 (Adair-Kirk et al., 1999
)
and the mammalian kidney AE1 variants
(Brosius et al., 1989
;
Kollert-Jons et al., 1993
;
Kudrycki and Shull, 1993
).
This peptide matches the sequence motif, YXX
, where X is any amino acid
and
is a hydrophobic residue. This motif associates with adaptor
complexes (Ohno et al., 1996
;
Dell'Angelica et al., 1997
;
Aguilar et al., 2001
;
Boehm and Bonifacino, 2001
) and
functions as an endocytic (Collawn et al.,
1990
) and trans-Golgi network (TGN) recycling
(Wong and Hong, 1993
) signal
for several membrane proteins. The AE1-3 variant and AE1-4 mutants that lack
this tyrosine-dependent sorting signal are rapidly degraded in MDCK cells,
suggesting that the trafficking events directed by this signal are necessary
for the stable accumulation of AE1 in this epithelial cell type.
To further define the sequence requirements for the sorting and cytoskeletal binding activities associated with the AE1-4 variant in transfected MDCK cells, we have fused various portions of the N-terminal cytoplasmic tail of AE1-4 to a cytoplasmic tailless version of the murine IgG FcRII B2 receptor. Studies with these chimeras revealed that multiple sorting activities reside within amino acids 1-63 of AE1-4. Amino acids 38-63 of AE1-4 were sufficient to direct efficient basolateral sorting of a chimeric receptor in MDCK cells. This region of AE1-4 was also sufficient to direct chimeric receptors from the basolateral membrane to the TGN. Another activity, which targeted chimeric receptors from the cell surface to late endosomes, was characterized within amino acids 1-37 of AE1-4. This region of AE1-4 was also capable of directing basolateral sorting. In addition to these sorting signals, amino acids 38-63 of AE1-4 were sufficient to mediate the association of chimeric receptors with the actin cytoskeleton. The alternative localization patterns exhibited by AE1/Fc receptor chimeras containing various combinations of these cytoplasmic activities suggest that interplay between these activities is critical for specifying the intracellular distribution of AE1-4 in epithelial cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Various portions of the N-terminal cytoplasmic tail of AE1-4 were amplified
by the polymerase chain reaction (PCR) and fused to the cytoplasmic tailless
Fc receptor that was cloned in the pcDNA3 mammalian expression
vector (Invitrogen). Sense oligonucleotides corresponding to nucleotides 26-40
or 137-151 of the AE1-4 anion exchanger cDNA
(Adair-Kirk et al., 1999) were
generated. These oligonucleotides were flanked at their 5' ends by an
AflII restriction site. Antisense oligonucleotides corresponding to
nucleotides 122-136 or 200-214 that were flanked at their 3' ends by a
stop codon followed by a XbaI site were also generated. These
oligonucleotides were used in various combinations to amplify the regions of
AE1-4 illustrated in the chimeras in Fig.
1. cDNAs encoding wild-type AE1-4, AE1-4Y44A, AE1-4Y47A or
AE1-4Y44AY47A (Adair-Kirk et al.,
1999
) were used as templates for these PCR reactions. For certain
reactions, antisense oligonucleotides that introduced an alanine for serine 25
or an alanine for leucine 50 were used for the amplifications. The PCR
reactions were performed using Pfu polymerase (Stratagene), and the
amplification products were digested with AflII and XbaI
restriction enzymes (Gibco) and ligated to the cytoplasmic tailless
Fc receptor utilizing the AflII site in
Fc- and an XbaI site in the polylinker of
pcDNA3. All chimeras were confirmed by DNA sequence analysis.
Cell culture and stable transfection
MDCK cells were maintained in Dulbecco's modified Eagle's media (DMEM)
supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin
and 100 µg/ml streptomycin at 37°C in 5% CO2. Some analyses
were performed with MDCK cells transiently expressing AE1/Fc
receptor chimeras that were introduced into the cells using the lipid-based
transfection reagent, Effectene (Qiagen). Alternatively, cell lines stably
expressing AE1/Fc receptor chimeras were established by growing
MDCK cells that were transfected by the calcium-phosphate method in the
presence of 600 µM G418. In all instances, similar results were observed in
stably and transiently transfected cells.
Immunolocalization analysis
Cells grown either on coverslips or on Transwell filters (Costar) were
washed in phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in
PBS and permeabilized by incubation in acetone. Following permeabilization,
the cells were washed with PBS and incubated with the rat 2.4G2
anti-Fc receptor monoclonal antibody
(Matter et al., 1992). After
washing, the cells were incubated with donkey anti-rat IgG conjugated to
lissamine (Jackson Immunoresearch) and phalloidin conjugated to fluorescein
isothiocyanate (FITC). Alternatively, cells were incubated with the rat
anti-Fc receptor monoclonal antibody and either a rabbit polyclonal
antibody directed against furin (Affinity Bioreagents) or a mouse monoclonal
antibody directed against the cation-independent mannose 6-phosphate receptor
(Affinity Bioreagents). Following washing, the cells were incubated with
donkey anti-rat IgG conjugated to lissamine, and either donkey anti-rabbit IgG
conjugated to FITC (Jackson Immunoresearch) or goat anti-mouse IgG conjugated
to FITC (Jackson Immunoresearch). In each instance, the cells were washed in
PBS and the localization of fluorescently labeled proteins was visualized
using either a Zeiss LSM 510 laser scanning microscope or a Zeiss Axiophot
epifluorescent microscope.
Cell-surface binding and internalization assays
MDCK cells stably expressing AE1/Fc receptor chimeras were grown
on coverslips or Transwell filters. In each instance, the cells were incubated
with the anti-Fc receptor antibody, which recognizes an
extracellular epitope of the receptor, for 1 hour at 4°C. Following
extensive washing with cold DMEM to remove unbound antibody, pre-warmed media
containing 5% fetal calf serum was added to the cells and they were incubated
for various times at 37°C. At each time point, the cells were fixed by
incubation in PBS containing 3% paraformaldehyde and permeabilized with PBS
containing 0.5% Triton X-100 (PBST). The cells were then incubated with the
rabbit anti-furin or the mouse anti-mannose 6-phosphate receptor antibodies in
PBST. The cells were again washed and incubated with donkey anti-rat IgG
conjugated to lissamine and either donkey anti-rabbit IgG conjugated to FITC
or goat anti-mouse IgG conjugated to FITC. Following washing, immunoreactive
polypeptides were visualized on a Zeiss LSM 510 laser-scanning microscope.
Experiments were performed to control for the possibility that the immunofluorescence profiles observed in internalization assays resulted from the dissociation of the Fc receptor antibody from the AE1/Fc chimeras following endocytosis. MDCK cells expressing the chimeras were incubated with the Fc receptor antibody as described above and shifted to 37°C for 45 minutes. The cells were then fixed and permeabilized and incubated with goat anti-rat Fab fragments conjugated to lissamine (Jackson Immunoresearch). Following washing, the cells were incubated with the Fc receptor antibody directly conjugated to Alexa 488 (Molecular Probes) to label the entire cellular pool of the chimera. The cells were again washed and immunoreactive polypeptides were visualized by confocal microscopy. This analysis revealed that the fluorescence derived from surface-labeled chimeras only accumulated in intracellular compartments that were also labeled by the directly conjugated Fc receptor antibody (data not shown), strongly suggesting that surface-bound antibodies did not dissociate from the chimeras following internalization into the cell.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
MDCK cells expressing a chimera that fused amino acids 1-63 of AE1-4 to this cytoplasmic tailless Fc receptor (FcR1-63, Fig. 2B) were fixed and double stained with a Fc-receptor-specific antibody and phalloidin. Confocal analysis revealed that this chimera primarily accumulated in the basolateral membrane and to a lesser extent in an intracellular membrane compartment of polarized MDCK cells. This result indicated that amino acids between 1 and 63 of AE1-4 are sufficient to direct basolateral sorting in this epithelial cell type.
Efficient basolateral sorting of AE1-4 in MDCK cells is dependent upon the
cytoplasmic tyrosine residues at amino acids 44 and 47
(Adair-Kirk et al., 1999). To
investigate the role of these residues in the basolateral sorting of
Fc1-63, point mutants were generated that substituted an alanine
for each of the tyrosine residues in this chimera separately or together
(Fig. 1). Substituting an
alanine for tyrosine 44, Fc1-63Y44A, resulted in a chimera that
still primarily accumulated in the basolateral membrane of transfected cells
(Fig. 2C). By contrast,
substituting an alanine for tyrosine 47, Fc1-63Y47A, resulted in a
polypeptide that accumulated both in the basolateral and apical membrane
(Fig. 2D), whereas the double
mutant, Fc1-63Y44AY47A, accumulated exclusively in the apical
membrane of transfected cells (Fig.
2E). These data indicated that tyrosines 44 and 47 were necessary
for the basolateral sorting activity that resides within amino acids 1-63 of
AE1-4. Furthermore, the results suggested that tyrosine 47, which is located
within the sequence YVEL (underlined in
Fig. 1), was critical for
efficient basolateral sorting of this chimera. Experiments described below
will further address the activities associated with the YVEL tetrapeptide.
The FcR1-37 and FcR38-63 chimeras are delivered
to the plasma membrane and recycled to distinct intracellular membrane
compartments
The acquisition of mature N-linked sugars by newly synthesized AE1-4 anion
exchangers is dependent upon recycling of these transporters from the plasma
membrane to the Golgi (Adair-Kirk et al.,
1999). The mutant anion exchangers, AE1-4
37 and AE1-4Y47A,
are partially defective in this recycling process
(Adair-Kirk et al., 1999
),
suggesting that the sequence between amino acids 1-37 and tyrosine 47 of AE1-4
are both necessary for efficient internalization from the plasma membrane and
subsequent Golgi recycling. Interestingly, chimeric receptors containing amino
acids 1-37, Fc1-37, or 38-63, Fc38-63
(Fig. 1), of AE1-4 accumulated
in intracellular membrane compartments in transfected MDCK cells
(Fig. 3). Confocal analyses
revealed that Fc1-37 accumulated in membrane vesicles that
partially overlapped the distribution of the cation-independent mannose
6-phosphate receptor, which primarily accumulates in late endosomes
(Fig. 3A-C). By contrast,
Fc38-63 was restricted to a perinuclear membrane compartment that
substantially overlapped the distribution of the TGN marker, furin
(Fig. 3D-F). Additional
experiments revealed that there was no overlap in the localization profiles of
Fc1-37 and furin, nor was there any significant overlap in the
localization profiles of Fc38-63 and the mannose 6-phosphate
receptor (data not shown). The observed accumulation of Fc1-37 and
Fc38-63 in intracellular membrane compartments may have been the
result of their specific retention in these compartments. Alternatively, amino
acids 1-37 and 38-63 of AE1-4 may each be able to direct the surface delivery
and subsequent recycling of these chimeric proteins to these intracellular
compartments.
|
To distinguish between these possibilities, internalization assays were
performed. Subconfluent cells expressing the chimeras were incubated with the
Fc receptor antibodies, which recognize an extracellular epitope on
the receptor. The incubation was carried out at 4°C to prevent
endocytosis. The cells were then shifted to 37°C, and the fate of
surface-labeled chimeras was followed over time by confocal microscopy. Prior
to shifting to 37°C, labeled Fc1-37 and Fc38-63 were
present on the cell surface where they did not overlap markers for
intracellular membrane compartments (data not shown). Although some of the
surface-labeled Fc1-37 was internalized from the plasma membrane 15
minutes after the shift to 37°C, a substantial fraction of
Fc1-37 still resided on the cell surface at this time point (arrows
in Fig. 4A). By 45 minutes,
however, the majority of surface-labeled Fc1-37 had internalized
and accumulated in a compartment that significantly overlapped the
distribution of the mannose 6-phosphate receptor
(Fig. 4). Unlike
Fc1-37, the bulk of surface-labeled Fc38-63 had
internalized 15 minutes after the shift to 37°C and its localization
partially overlapped the distribution of the TGN marker, furin
(Fig. 5). Forty-five minutes
after the shift, the localization profiles of Fc38-63 and furin
were very similar (Fig. 5).
These data indicated that sequences between amino acids 1-37 and 38-63 of
AE1-4 can independently direct the internalization of chimeric proteins from
the cell surface. These results may account for the observation that
AE1-437 and AE1-4Y47A are only partially defective in recycling, since
one internalization signal is presumably functional in each.
|
|
The internalization of Fc1-37 and Fc38-63 from the cell surface was not induced by antibody binding to the chimeras, since studies with Fc1-63 revealed that this chimera remained on the cell surface throughout the time course of similar assays (data not shown). Although surface labeled Fc1-63 did not internalize, it did undergo redistribution on the cell surface to sites of cell-cell contact. This redistribution of Fc1-63 mimicked the redistribution of phalloidin-stained microfilaments in these cells, suggesting a critical role for the actin cytoskeleton in maintaining the cell-surface localization of this chimeric receptor.
Identification of amino acids necessary for the sorting activities
associated with amino acids 1-37 and 38-63 of AE1-4
Internalization assays performed with polarized MDCK cells expressing
Fc38-63 yielded results essentially identical to those shown in
Fig. 5 (data not shown).
However, incubation of the Fc receptor antibody at 4°C with the
apical and basolateral surfaces of cells grown on permeable supports revealed
that the surface population of Fc38-63 accumulated exclusively in
the basolateral membrane of this polarized epithelial cell type
(Fig. 6A). This result
indicates that sequences within amino acids 38-63 of AE1-4 are not only
sufficient to direct internalization from the plasma membrane and subsequent
transport to the TGN, but are also sufficient to direct basolateral sorting in
MDCK cells. The fact that tyrosines 44 and 47 of AE1-4 are necessary for both
the basolateral sorting and Golgi recycling activities of this transporter in
MDCK cells (Adair-Kirk et al.,
1999) prompted us to examine the role of these residues in the
trafficking of Fc38-63. Mutants were generated that substituted an
alanine for each of the tyrosines in this chimera. MDCK cells expressing the
mutant chimeras were fixed and stained with the Fc receptor
antibody and phalloidin. This analysis revealed that Fc38-63Y44A
(data not shown) exhibited a steady-state pattern of localization
indistinguishable from Fc38-63
(Fig. 6B). Longer exposure of
the image in Fig. 6B again
revealed that the surface population of Fc38-63 was exclusively
basolateral (data not shown). By contrast, Fc38-63Y47A accumulated
in both the basolateral and apical membranes of polarized MDCK cells and to a
lesser extent in an intracellular membrane compartment
(Fig. 6C). This result
suggested that tyrosine 47 was critical both for efficient basolateral sorting
and for efficient endocytosis of Fc38-63 following cell-surface
delivery. To directly test whether tyrosine 47 was critical for endocytosis,
internalization assays identical to those described above were performed with
Fc38-63Y47A. This analysis revealed that surface-labeled
Fc38-63Y47A was almost entirely retained in the plasma membrane
throughout the time course of the assay (data not shown), illustrating the
importance of this residue for efficient endocytosis. This result provides
additional evidence that the uptake of surface-labeled chimeras in our
internalization assay does not simply occur as a consequence of antibody
binding. Furthermore, the very slow rate of internalization of
Fc38-63Y47A suggests that the surface distribution of this chimera
primarily resulted from sorting events that occurred at the level of the
TGN.
|
The hydrophobic residue in the YXX motif is critical for the ability
of this motif to serve as a sorting signal
(Collawn et al., 1990
;
Wong and Hong, 1993
) and for
its ability to associate with adaptins
(Ohno et al., 1996
;
Dell'Angelica et al., 1997
;
Aguilar et al., 2001
;
Boehm and Bonifacino, 2001
). To
assess the contribution of the leucine residue in the YVEL50
sequence of AE1-4 (Fig. 1) to
the sorting activities contained within amino acids 38-63, we mutated this
residue to an alanine. This amino-acid substitution did not alter the
basolateral sorting activity of Fc38-63, as the surface population
of Fc38-63L50A primarily accumulated in the basolateral membrane of
polarized MDCK cells (Fig. 6E).
Although this chimera could still be detected in a sub-apical compartment of
transfected cells, the majority of Fc38-63L50A resided on the cell
surface. The fact that efficient basolateral sorting was unaffected by
substituting an alanine for leucine 50, whereas the accumulation of this
chimera in intracellular compartments was substantially reduced, suggested
that there were different sequence requirements for the basolateral sorting
and endocytic activities of Fc38-63. However, it must be pointed
out that the reduced intracellular accumulation of Fc38-63L50A
could be due to a reduced rate of endocytosis as well as an increased rate of
recycling to the basolateral membrane following internalization. Whether one
or both of these possibilities contributes to the localization of this chimera
in MDCK cells is not known.
The data described above suggested that the YVEL peptide of AE1-4 comprises
a YXX motif that is critical for the sorting activities associated with
this transporter. The fact that alanine is a relatively hydrophobic residue in
many of the algorithms used to predict hydrophobicity further suggested the
possibility that the residual basolateral sorting activity associated with
Fc38-63Y47A (Fig.
6C) resulted from the creation of a YXX
sequence surrounding
tyrosine 44. To test whether a more hydrophobic residue at amino acid 47 would
fully suppress the sorting defects associated with Fc38-63Y47A, we
replaced tyrosine 47 with a leucine. Confocal analysis revealed that this
mutant, Fc38-63Y47L (Fig.
6D), exhibited a localization profile very similar to
Fc38-63Y47A (Fig.
6C). The failure of this mutant to undergo efficient basolateral
sorting and subsequent internalization from the plasma membrane demonstrates
that the sequence surrounding the tyrosine and leucine residues in the YXXL
tetrapeptide of AE1-4 is critical for determining its sorting activity.
As was observed for Fc38-63, internalization assays with
polarized MDCK cells expressing Fc1-37 yielded results very similar
to those shown in Fig. 4 (data
not shown). Surprisingly, incubation of the Fc receptor antibody at
4°C with the apical and basolateral surfaces of cells revealed that a
significant percentage of the surface population of Fc1-37
accumulated in the basolateral membrane of polarized cells
(Fig. 7A). This suggested that
amino acids 1-37 of AE1-4 contained an inefficient basolateral sorting signal,
as well as sequences sufficient to direct the internalization of chimeric
receptors from the cell surface to late endosomes. A comparison of the
sequence within this region to other known sorting signals revealed that amino
acids 17-25 (underlined in Fig.
1) are similar to a cytoplasmic sorting signal that is involved in
both the basolateral sorting and transcytosis of the poly Ig receptor
(Casanova et al., 1990;
Casanova et al., 1991
). The
residues in Fig. 1 that are
marked with a plus are identical to amino acids in this poly Ig receptor
signal. The serine in the poly Ig receptor signal is required for the
transcytosis of the receptor from the basolateral to the apical membrane of
transfected MDCK cells (Casanova et al.,
1990
). To determine whether the serine in the putative sorting
signal in Fc1-37 was involved in directing the intracellular
trafficking of this chimera, a mutant was generated that substituted an
alanine for serine, Fc1-37S25A. Immunolocalization analyses
revealed that unlike the steady-state localization profile of
Fc1-37 (Fig. 7B),
Fc1-37S25A accumulated exclusively in the apical membrane of
transfected cells (Fig. 7C).
This pattern of localization suggested that serine 25 was necessary for both
the basolateral sorting and endocytic activities that resided within amino
acids 1-37. Again, to directly test the role of this residue in endocytosis,
internalization assays were performed with Fc1-37S25A. This
analysis indicated that the surface population of this chimera was retained in
the apical membrane during the time course of the assay (data not shown),
demonstrating that serine 25 is critical for efficient endocytosis.
|
Similar sequence requirements for the basolateral sorting and the
cytoskeletal association of AE1-4 in MDCK cells
Previous analyses had shown that AE1-4 colocalizes both with actin stress
fibers underlying the basal membrane of subconfluent MDCK cells and with
cortical actin at sites of cell-cell contact
(Adair-Kirk et al., 1999). To
determine whether the AE1/Fc chimeras that accumulate in the
basolateral membrane of polarized MDCK cells exhibit a similar capacity to
colocalize with elements of the actin cytoskeleton, subconfluent MDCK cells
expressing Fc1-63 (Fig.
8A-C) were double stained with phalloidin and the
Fc-receptor-specific antibody. This analysis revealed that
Fc1-63 colocalized both with actin stress fibers and with cortical
actin at sites of cell-cell contact. Similar analyses with
Fc38-63Y47A, which inefficiently sorts to the basolateral membrane
of MDCK cells (Fig. 6C),
revealed that this chimera also colocalized with stress fibers and with
cortical actin (Fig. 8D-F).
This result indicated that sequences between amino acids 38 and 63 of AE1-4
are not only sufficient to direct basolateral sorting, they can also mediate
the association of a chimeric receptor with various elements of the actin
cytoskeleton. Whether this interaction represents a functional association
with actin that is involved in the basolateral sorting of AE1-4 in MDCK cells
is not known.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequences within amino acids 38-63 of AE1-4 are sufficient to mediate the association of a chimeric receptor with actin stress fibers in subconfluent MDCK cells. Yet Fc38-63, which is sorted to the basolateral membrane of polarized cells, is rapidly internalized and delivered to the TGN. If indeed the surface retention of Fc1-63 is due to its interaction with actin, the observation that Fc38-63 is rapidly internalized following surface delivery suggests that sequences within amino acids 1-37 of AE1-4 are necessary for a stable association with actin. This idea is supported by the observation that some of the surface population of Fc1-37 colocalizes with actin stress fibers in the basal membrane of polarized MDCK cells (data not shown). In the absence of this cytoskeletal binding activity within amino acids 1-37, the endocytic activity of the Y47VEL peptide is dominant and it directs the rapid internalization of Fc38-63. Mutations in the YVEL peptide that reduce its endocytic activity, such as the tyrosine 47 to alanine substitution, results in a chimera that is primarily retained on the cell surface where it colocalizes with actin.
Other investigators have shown that certain membrane proteins, including
TGN38 (Wong and Hong, 1993)
and furin (Schafer et al.,
1995
), recycle from the plasma membrane to the TGN. Each of these
proteins contains a YXXL tetrapeptide in their cytoplasmic domain, and in the
case of TGN38, this sequence is necessary to direct recycling to the TGN
(Wong and Hong, 1993
).
Furthermore, both the tyrosine and leucine residues are necessary for the
recycling activity of TGN38. Our chimera studies have revealed a similar role
for the YVEL peptide of AE1-4 in TGN recycling. Mutation of either the
tyrosine or leucine residue in this sequence to an alanine dramatically
increases the percentage of the Fc38-63 chimera that resides on the
cell surface. By contrast, the basolateral sorting activity of
Fc38-63 is substantially inhibited by mutation of the tyrosine
residue and unaffected by mutation of the leucine. The differential effect of
these mutations on basolateral sorting and endocytosis suggest distinct
requirements for the recognition of this tyrosine-based signal by the sorting
machinery at the TGN and plasma membrane.
Our studies with Fc1-37 have shown that amino acids 1-37 of
AE1-4 possess an endocytic activity as well as a weak basolateral sorting
signal. The fact that Fc1-63Y44A, Y47A accumulates exclusively in the apical
membrane of polarized MDCK cells (Fig.
2E) suggests that the basolateral sorting activity within amino
acids 1-37 is masked or non-functional in this context. Although the basis for
this is not understood, previous analyses have shown that mutation of the
tyrosine-dependent basolateral sorting signal of the full-length AE1-4
variant, AE1-4Y44A, Y47A, does not completely abrogate the basolateral sorting
of this membrane transporter (Adair-Kirk et
al., 1999). At this time it is not clear whether the residual
basolateral accumulation of AE1-4Y44A, Y47A is dependent upon the signal
within amino acids 1-37.
Amino acids 17-25 of AE1-4 (Fig.
1) share sequence similarity with the membrane proximal
cytoplasmic sorting signal of the poly Ig receptor. Other investigators have
shown that the serine residue in the poly Ig receptor signal, which is
homologous to serine 25 in AE1-4, is necessary for the transcytosis of the
receptor (Casanova et al.,
1990). In addition, the histidine and arginine in the poly Ig
receptor signal, which are homologous to amino acids 17 and 18, respectively,
in AE1-4, are necessary for basolateral sorting of the receptor
(Casanova et al., 1991
). Our
mutagenesis studies with AE1/Fc chimeras have indicated that serine
25 is necessary for both the endocytic and basolateral sorting activities that
reside within amino acids 1-37 of AE1-4. It is not known whether histidine 17
and arginine 18 of AE1-4 are necessary for either of the sorting activities
associated with amino acids 1-37. However, additional studies have shown that
AE1-4
21Y47A accumulates in the apical membrane of MDCK cells (data not
shown), whereas AE1-4Y47A accumulates in the basolateral membrane
(Adair-Kirk et al., 1999
). This
suggests that residues in the first 21 amino acids of AE1-4 contribute to the
basolateral sorting of this electroneutral transporter in this epithelial cell
type.
Previous pulse-chase studies have shown that deletion of the N-terminal 37
amino acids of AE1-4 dramatically slows the rate at which this polypeptide
recycles to the Golgi for the acquisition of mature N-linked sugars. Yet at
steady state, the ratio of AE1-437 with mature to immature N-linked
sugars is not significantly different from the wild-type AE1-4
(Adair-Kirk et al., 1999
). By
contrast, even though AE1-4Y47A recycles much more rapidly than
AE1-4
37, the majority of AE1-4Y47A in the cell possesses immature
N-linked sugars (Adair-Kirk et al.,
1999
). One explanation that could account for these observations
is that the late endosomal targeting signal that we have characterized within
amino acids 1-37 is involved in regulating the constitutive turn over of AE1-4
in this epithelial cell type. In the absence of this sorting signal,
AE1-4
37 turns over slowly. As a consequence of this slower turnover,
newly synthesized polypeptides have more time to recycle to the Golgi and
acquire mature N-linked sugars. This would result in a steady-state profile
for AE1-4
37 that is similar to wild-type AE1-4 even though the two
proteins recycle at very different rates. The observation that AE1-4Y47A with
mature N-linked sugars does not accumulate in the cell may be a result of the
sorting signal within amino acids 1-37 directing a more rapid rate of turnover
of this polypeptide in the absence of a wild-type tyrosine-dependent signal.
Future studies will address how the interplay between the sorting signals we
have characterized at the N-terminus of AE1-4 controls the Golgi recycling and
stability of this variant transporter in MDCK cells.
Studies of investigators have shown that the actin cytoskeleton is involved
in regulating the vesicular transport
(Rozelle et al., 2000;
Brown and Song, 2001
;
Kanzaki et al., 2001
;
Valderrama et al., 2001
) and
endocytosis (Fujimoto et al.,
2000
; Pol et al.,
2000
; Jiang et al.,
2002
) of some membrane proteins. Our data presented here as well
as previous analyses (Adair-Kirk et al.,
1999
) suggest that the actin cytoskeleton plays a critical role in
directing the localization of AE1 in polarized MDCK cells. Although it is
unclear whether the ability to associate with actin is directly involved in
the Golgi recycling or turnover of AE1-4, mechanisms must exist to regulate
the availability of the sorting signals at the N-terminus of AE1-4 to elements
of the cellular sorting machinery. In the case of the poly Ig receptor, it has
been proposed that the phosphorylation of the serine residue within its
cytoplasmic sorting signal is required for the transcytosis of the receptor
(Casanova et al., 1990
). It is
tempting to speculate that similar phosphorylation events may be involved in
determining how the various activities at the N-terminus of AE1-4 are
coordinated to regulate its localization and stability. Along these lines it
is interesting to note that the tyrosine residue in skate erythroid AE1 that
is homologous to tyrosine 47 in chicken AE1-4 is phosphorylated
(Musch et al., 1999
). In
addition, the phosphorylation of this residue is stimulated when skate
erythroid cells are grown in hypertonic medium. Whether tyrosine 47 or other
residues at the N-terminus of AE1-4 are phosphorylated and the potential role
of these phosphorylation events in regulating AE1 localization in epithelial
cells will be the subject of future investigations.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adair-Kirk, T. L., Cox, K. H. and Cox, J. V.
(1999). Intracellular trafficking of variant chicken kidney AE1
anion exchangers: Role of the alternative N-terminal sequences in polarized
sorting and Golgi recycling. J. Cell Biol.
147,1237
-1248.
Aguilar, R. C., Boehm, M., Gorshkova, I., Crouch, R. J., Tomita,
K., Saito, T., Ohno, H. and Bonifacino, J. S. (2001).
Signal-binding specificity of the mu4 subunit of the adaptor protein complex
AP-4. J. Biol. Chem.
276,13145
-13152.
Boehm, M. and Bonifacino, J. S. (2001).
Adaptins: the final recount. Mol. Biol. Cell
12,2907
-2920.
Brosius, F. C., Alper, S. L., Garcia, A. M. and Lodish, H.
F. (1989). The major kidney band 3 gene transcript predicts
an amino-terminal truncated band 3 polypeptide. J. Biol.
Chem. 264,7784
-7787.
Brown, B. K. and Song, W. (2001). The actin cytoskeleton is required for the trafficking of the B cell antigen receptor to the late endosomes. Traffic 2, 414-427.[CrossRef][Medline]
Casanova, J. E., Breitfeld, P. P., Ross, S. A. and Mostov, K. E. (1990). Phosphorylation of the polymeric immunoglobulin receptor is required for its efficient transcytosis. Science 248,742 -745.[Medline]
Casanova, J. E., Apodaca, G. and Mostov, K. E. (1991). An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell 66,65 -75.[Medline]
Collawn, J. F., Stangel, M., Kuhn, L. A., Esekogwu, V., Jing, S. Q., Trowbridge, I. S. and Tainer, J. A. (1990). Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell 63,1061 -1072.[Medline]
Dell'Angelica, E. C., Ohno, H., Ooi, C. E., Rabinovich, E.,
Roche, K. W. and Bonifacino, J. S. (1997). AP-3: an
adaptor-like protein complex with ubiquitous expression. EMBO
J. 16,917
-928.
Fujimoto, L. M., Roth, R., Heuser, J. E. and Schmid, S. L. (2000). Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic 1,161 -171.[CrossRef][Medline]
Ghosh, S., Cox, K. H. and Cox, J. V. (1999).
Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during
recycling to the Golgi. Mol. Biol. Cell
10,455
-469.
Jiang, Z. Y., Chawla, A., Bose, A., Way, M., Czech and M. P.
(2002). A phosphatidylinositol 3-kinase-independent insulin
signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose
transporter recycling. J. Biol. Chem.
277,509
-515.
Kanzaki, M., Watson, R. T., Khan, A. H. and Pessin, J. E.
(2001). Insulin stimulates actin comet tails on intracellular
GLUT4-containing compartments in differentiated 3T3L1 adipocytes.
J. Biol. Chem. 276,49331
-49336.
Kollert-Jons, A., Wagner, S., Hubner, S., Appelhans, H. and
Drenckhahn, D. (1993). Anion exchanger 1 in human kidney and
oncocytoma differs from erythroid AE1 in its NH2 terminus.
Am. J. Physiol. 265,F813
-F821.
Kudrycki, K. E. and Shull, G. E. (1993). Rat kidney band 3 chloride/bicarbonate exchanger mRNA is transcribed from an alternative promoter. Am. J. Phys. 264,F540 -F547.
Matter, K., Hunziker, W. and Mellman, I. (1992). Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71,741 -753.[Medline]
Musch, M. W., Hubert, E. M. and Goldstein, L.
(1999). Volume expansion stimulates p72(syk) and p56(lyn) in
skate erythrocytes. J. Biol. Chem.
274,7923
-7928.
Nelson, W. J. and Hammerton, R. W. (1989). A membrane cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in MDCK cells: Implications for the biogenesis of epithelial cell polarity. J. Cell Biol. 108,893 -902.[Abstract]
Ohno, H., Fournier, M. C., Poy, G. and Bonifacino, J. S.
(1996). Structural determinants of interaction of tyrosine-based
sorting signals with the adaptor medium chains. J. Biol.
Chem. 271,29009
-29015.
Pol, A., Lu, A., Pons, M., Peiro, S. and Enrich, C.
(2000). Epidermal growth factor-mediated caveolin recruitment to
early endosomes and MAPK activation. Role of cholesterol and actin
cytoskeleton. J. Biol. Chem.
275,30566
-30572.
Rozelle, A. L., Machesky, L. M., Yamamoto, M., Driessens, M. H., Insall, R. H., Roth, M. G., Luby-Phelps, K., Marriott, G., Hall, A. and Yin, H. L. (2000). Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3. Curr. Biol. 10,311 -320.[CrossRef][Medline]
Schafer, W., Stroh, A., Berghofer, S., Seiler, J., Vey, M., Kruse, M. L., Kern, H. F., Klenk, H. D. and Garten, W. (1995). Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J. 14,2424 -2435.[Abstract]
Valderrama, F., Duran, J. M., Babia, T., Barth, H., Renau-Piqueras, J. and Egea, G. (2001). Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells. Traffic 2, 717-726.[CrossRef][Medline]
Wong, S. H. and Hong, W. (1993). The SXYQRL
sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi
network localization. J. Biol. Chem.
268,22853
-22862.
Yoo, J. S., Moyer, B. D., Bannykh, S., Yoo, H. M., Riordan, J.
R. and Balch, W. E. (2002). Non-conventional trafficking of
the cystic fibrosis transmembrane conductance regulator through the early
secretory pathway. J. Biol. Chem.
277,11401
-11409.