From the Zentrum für Molekulare Neurobiologie Hamburg, Hamburg University, Falkenried 94, D-20246 Hamburg, Germany
Received for publication, November 26, 2000
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ABSTRACT |
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The ClC-5 chloride channel resides mainly in
vesicles of the endocytotic pathway and contributes to their
acidification. Its disruption in mice entails a broad defect in renal
endocytosis and causes secondary changes in calciotropic hormone
levels. Inactivating mutations in Dent's disease lead to proteinuria
and kidney stones. Possibly by recycling, a small fraction of ClC-5
also reaches the plasma membrane. Here we identify a
carboxyl-terminal internalization motif in ClC-5. It resembles
the PY motif, which is crucial for the endocytosis and degradation of
epithelial Na+ channels. Mutating this motif
increases surface expression and currents about 2-fold. This is
probably because of interactions with WW domains, because dominant
negative mutants of the ubiquitin-protein ligase WWP2 increased surface
expression and currents of ClC-5 only when its PY motif was intact.
Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L
mutant decreased WT ClC-5 currents but did not affect channels with
mutated motifs. Similarly, decreasing endocytosis by expressing the
inactive S34N mutant of rab5 increased ClC-5 currents only if its
PY-like motif was intact. Thus, the endocytosis of ClC-5, which itself
is crucial for the endocytosis of other proteins, depends on the
interaction of a carboxyl-terminal internalization signal with
ubiquitin-protein ligases containing WW domains.
CLC chloride channels form a gene family of chloride channels that
has at least nine members in mammals (1). Based on homology, it can be
divided into three branches. Channels of the first branch perform their
physiological function in the plasma membrane. In some instances, this
conclusion is also supported by inherited disease. Mutations in
the ClC-1 chloride channel cause myotonia (2, 3) because its
conductance is essential for the electrical stability of the muscle
plasma membrane. Mutations in the human ClC-Kb kidney chloride channel
are a cause of Bartter's syndrome (4), and disruption of the mouse
ClC-K1 leads to renal diabetes insipidus (5). Both findings indicate
that these channels are involved in the transport of salt and fluid
across different nephron segments.
In contrast, members of the two remaining branches of the CLC gene
family probably function primarily in intracellular compartments (1).
Again, an inherited disease has shed light on one of these channels.
ClC-5, which belongs to the branch also comprising ClC-3 and ClC-4, is
mutated in Dent's disease (6). This X-linked disorder is characterized
by the urinary loss of low molecular weight proteins, calcium, and
phosphate, and leads to the clinically important symptoms of kidney
stones and nephrocalcinosis. The selective loss of low molecular weight
proteins points to a defect of the renal proximal tubule of the kidney,
where filtered proteins are normally reabsorbed by endocytosis. Indeed,
ClC-5 is located in subapical endosomes of the proximal tubule where it
colocalizes with the V-type H+-ATPase and with reabsorbed
protein (7, 8). In transfected cells, besides some labeling of the
plasma membrane, ClC-5 was mainly found in intracellular vesicles,
where it colocalizes with endocytosed protein (7). Mice with a
disrupted ClC-5 gene indeed had a broad defect in proximal tubular
endocytosis (9). Fluid-phase endocytosis, receptor-mediated
endocytosis, and the endocytotic removal of plasma membrane proteins
were markedly inhibited but not totally abolished. Because several
hormones or their precursors are endocytosed from the primary urine,
this led to secondary changes in calciotropic hormones and changes in
phosphate excretion (9). ClC-5 probably provides an electric shunt for
the acidification of endosomes (7). Indeed, preliminary experiments
revealed that renal endosomes isolated from ClC-5 knockout mice were
acidified at slower rates (9).
When ClC-5 was expressed in Xenopus oocytes (10, 11) or in
mammalian cells (10), it induced strongly rectifying chloride currents
that were detectable only at voltages more positive than +20 mV. Point
mutations that changed the voltage dependence, ion selectivity, and
rectification demonstrated that ClC-5 directly mediates plasma membrane
chloride currents (10). Thus, consistent with the immunocytochemistry
of transfected cells (7), a small fraction of ClC-5 also resides in the
plasma membrane.
The cytoplasmic carboxyl terminus of ClC-5 contains between its two
conserved CBS1 domains (12) a proline-rich stretch of
amino acids (PPLPPY). A similar sequence is not found in the otherwise
highly related ClC-3 and ClC-4 channels. This sequence bears
resemblance to the PY motif that was recognized in the human epithelial
Na+ channel (ENaC) (13). Each of
the three different subunits ( A peptide corresponding to the PY motif of ClC-5 was shown to bind to
several WW domains within the proteins WWP1, WWP2, and WWP3 (18). WW
domains are conserved protein motifs comprising ~35 amino acids. They
are characterized by two highly conserved tryptophan residues, a
central core of aromatic and hydrophobic residues, and a proline
located three residues downstream from the second tryptophan (19). WWP2
(and probably the other, partially characterized proteins as well)
belongs to the same protein class as Nedd4 (20) with which it shares
several domains: a C2 (Ca2+/lipid binding) domain, multiple
(three or four) WW domains, and a carboxyl-terminal ubiquitin-protein
ligase (HECT) domain.
Given the pivotal role of ClC-5 in renal endocytosis (7, 9), it is of
great interest to identify signals that are involved in ClC-5
trafficking. We therefore investigated whether the PY motif on ClC-5
plays a role in endocytosis of the channel protein. Indeed, disrupting
this motif by site-directed mutagenesis led to increased surface
retention and to higher currents. Coexpression with mutants of rab5 and
WWP2 indicate that the PY motif is in fact important for endocytosis, a
process probably depending on interactions with proteins of the Nedd4
family. Thus, we have identified a motif that is important for the
endocytosis of ClC-5, which itself plays an important role in
endocytotic processes (9).
Molecular Biology
ClC-5 and WWP2 Constructs--
These cDNAs were cloned into
pTLN (21), a vector containing Xenopus globin untranslated
sequences and which is optimized for protein expression in
Xenopus oocytes.
rab5 Constructs--
This cDNA was cloned into the
eukaryotic expression vector pFrog3 derived from pCDNA3
(Invitrogen) by flanking the multiple cloning site with the 5' and 3'
untranslated region from the Xenopus globin gene (7). rab5
constructs were amino-terminally tagged with the Myc epitope
(MEQKLISEEDLQS) (7). Point mutations were introduced by recombinant
polymerase chain reaction. All polymerase chain reaction-derived
fragments were entirely sequenced.
Expression in Xenopus laevis Oocytes and Voltage Clamp
Analysis
Capped cRNA was transcribed from constructs linearized by MluI
using the mMessage mMachine kit (Ambion) and SP6 polymerase (for
constructs in pTLN) or T7 polymerase (pFrog3 constructs). A total
amount of 10 ng of cRNA was injected into defolliculated oocytes,
i.e. 5 ng of cRNA each in coexpression studies. Oocytes were
kept at 17 °C in modified Barth's solution (88 mM NaCl,
2.4 mM NaHCO3, 1 mM KCl, 0.41 mM CaCl2, 0.33 mM
Ca(NO3)2, 0.82 mM MgSO4, 10 mM HEPES, 50 µg/ml tetracyclin, 20 µg/ml gentamycin, pH 7.6). Standard two-electrode voltage clamp
measurements were performed at room temperature 2-3 days after
injection using Turbotec 05 or 10C amplifiers (NPI Instruments, Tamm,
Germany) and pClamp 5.5 software (Axon Instruments, Foster City, CA).
Data are given as means ± S.E. Currents were recorded in
ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4).
Determination of Surface Expression
Surface expression of the ClC-5 protein was determined by the
method of Zerangue et al. (22). An HA epitope was inserted by polymerase chain reaction mutagenesis into the extracellular loop
between transmembrane domains D1 and D2 of wild type (WT) ClC-5 and
mutants. After 3 days at 17 °C, oocytes were placed for 30 min in
ND96 with 1% BSA at 4 °C to block unspecific binding, incubated for
60 min at 4 °C with 1 µg/ml rat monoclonal anti-HA antibody (3F10,
Roche Molecular Biochemicals, in 1% BSA/ND96), washed at 4 °C, and
incubated with horseradish peroxidase-coupled secondary antibody (goat
anti-rat Fab fragments, Jackson ImmunoResearch, in 1% BSA for 30-60
min at 4 °C). Oocytes were washed thoroughly (1% BSA, 4 °C, 60 min) and transferred to frog Ringer solution (82.5 mM NaCl,
2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.5) without BSA. Individual oocytes were
placed in 50 µl of Power Signal Elisa solution (Pierce) and incubated
at room temperature for 1 min. Chemiluminescence was quantified in a
Turner TD-20/20 luminometer (Turner Design, Sunnyvale, CA).
Western Blot Analysis
The oocytes used to measure the surface expression of channel
subunits were subsequently pooled and stored at Northern Blot Analysis
A human multiple tissue Northern blot of polyadenylated RNA (2 µg/tissue; CLONTECH) was hybridized under high
stringency conditions with a 32P-labeled cDNA probe
encompassing the entire open reading frame of human WWP2 using standard
conditions. Autoradiography used a phosphoimager (Fuji Bas-1500)
or photographic films.
The carboxyl terminus of ClC-5 carries a proline-rich stretch of
amino acids that shows similarity to a PY motif and that is not present
in the highly related ClC-3 and ClC-4 proteins (Fig.
1A). We mutated several
prolines and the tyrosine residue within the motif (Fig. 1B)
and measured the effect of these mutations on currents in the
Xenopus oocyte expression system. There was no change in
their macroscopic biophysical properties, like rectification (Fig.
1C) or ion selectivity (data not shown). However, these mutations consistently yielded approximately 2-fold higher currents (Fig. 1D).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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,
, and
) of this heteromeric
channel contains a carboxyl-terminal motif with the consensus sequence
PPXY. The motifs on the
- and
-subunits can interact
with Nedd4, a ubiquitin-protein ligase, by binding to WW domains of the
latter protein (14). This interaction leads to the ubiquitination of
ENaC (15) and to its internalization by clathrin-mediated endocytosis
(16) and to its degradation. This process is physiologically important,
because the distal tubular Na+ reabsorption mediated
by ENaC needs to be tightly regulated. Mutations that truncate or
disrupt the PY motif in the carboxyl termini of
or
ENaC
subunits lead to increased channel activity primarily due to a markedly
prolonged surface retention time. This explains the gain of sodium
channel activity observed in Liddle's syndrome, a rare and very severe
form of human inherited hypertension (15-17). Mutational analysis
revealed that the tyrosine residue of the PY motif is critical for the
recognition and endocytotic retrieval of ENaC from the plasma membrane
(16).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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20 °C. After homogenization of the pooled oocytes in an ice-cold solution containing 250 mM sucrose, 0.5 mM EDTA, 5 mM
Tris-HCl, pH 7.4, and a protease inhibitor mix (Complete®,
Roche Molecular Biochemicals), yolk platelets were removed by
three low speed centrifugation steps (500 × g, 2 min).
The resulting supernatant was mixed with Laemmli sample buffer, and the
protein equivalent to one oocyte was analyzed by SDS-polyacrylamide gel
electrophoresis (10% polyacrylamide). After transfer to
nitrocellulose, blots were blocked in Tris-buffered saline (150 mM NaCl, 25 mM Tris, pH 7.4) containing 5%
milk powder and 0.1% Nonidet P-40. Primary (rat anti-HA monoclonal
3F10; 200 ng/ml; Roche Molecular Biochemicals) and secondary
(horseradish peroxidase-conjugated goat anti-rat IgG; 1:10,000; Jackson
ImmunoResearch) antibodies were diluted in Tris-buffered saline
blocking solution. Washes were in Tris-buffered saline with 0.1%
Nonidet P-40. Reacting proteins were detected by using the Renaissance
reagent (PerkinElmer Life Sciences) and photographic films (Kodak).
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ABSTRACT
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DISCUSSION
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Fig. 1.
Mutational analysis of the PY motif of
ClC-5. A, comparison of the carboxyl-terminal regions
of ClC-3, ClC-4, and ClC-5. The two conserved CBS domains are
indicated, and the proline-rich PY motif present exclusively in ClC-5
is shown in bold. B, mutations of the ClC-5 PY
motif used in this study. C, I-V relationships of ClC-5,
ClC-5(AAE_A), and ClC-5(Y627E) currents (n = 10).
Steady-state currents measured by a two-electrode voltage clamp
of Xenopus oocytes are shown. D, bar diagram of
current levels obtained from WT and mutant ClC-5 at +80 mV
(n = 10; error bars indicate ± S.E.).
To investigate whether mutations in the PY motif enhance currents by
increasing the surface expression of the channel protein, we inserted
an HA epitope into the extracellular loop between D1-D2 of WT and the
two ClC-5 mutants. The tagged constructs yielded currents that were
indistinguishable from wild type (data not shown). The amount of HA
epitopes at the cell surface was quantified by incubating
nonpermeabilized oocytes with a monoclonal anti-HA antibody, followed
by an enzymatic amplification procedure that uses chemiluminescence as
the final step (22). As shown in Fig. 2A, the surface expression
correlated well with the current amplitudes measured in
Xenopus oocytes. To rule out effects on translation efficiency or overall protein stability, the total amount of ClC-5 was
determined by Western blotting extracts of the same oocytes used for
the surface expression assay. The ClC-5(AAE_A) mutant is expressed to
the same overall amount in oocytes as WT ClC-5 (Fig. 2B).
The effect of the ClC-5(AAE_A) mutants could be due to the replacement
of tyrosine by the negatively charged glutamate. This might mimic a
tyrosine phosphorylation. We therefore compared currents of the
ClC-5(Y672E) mutant with those of ClC-5(Y672A) but could not detect any
differences (data not shown), making this explanation unlikely.
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We next investigated the influence of the PY motif on the surface
retention time of ClC-5 under conditions in which the insertion of
newly synthesized channels into the plasma membrane is inhibited by
brefeldin A (BFA). BFA blocks the anterograde vesicular transport from
the endoplasmic reticulum and causes a redistribution of Golgi
cisternae to the endoplasmic reticulum (23). Although BFA has multiple
targets in vesicular transport, it does not affect clathrin-mediated
endocytosis in the plasma membrane (24). It is known that BFA is also
efficient in Xenopus oocytes (16, 25). Thus, in the presence
of BFA plasma membrane currents reflect the half-lives of plasma
membrane resident channels, without complications arising from newly
synthesized proteins. However, BFA treatment does not exclude the
possibility that endocytosed channels are recycled back to the
surface. Oocytes were injected with cRNA encoding WT and AAE_A mutant
ClC-5. When currents had reached steady-state levels after 3 days, BFA
was added to oocytes, and currents were determined over a period of
22 h. Wild type currents decreased with a half-time of about
23 h. In contrast, the decay of currents from the AAE_A mutant was
dramatically slowed (Fig. 3). Thus,
mutating the PY motif in ClC-5 markedly increases its stability in the
plasma membrane.
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ClC-5 was colocalized with markers of early endosomes (7, 8) and was
present in the large vesicles created by overexpressing the
GTPase-deficient Q79L mutant of rab5 (7). rab5 is an important regulator of endocytosis. Overexpression in Xenopus oocytes
of rab5 or its Q79L mutant increases fluid phase endocytosis, whereas the opposite is true for the dominant negative S34N mutant (26). To
test whether global changes in endocytosis affect ClC-5 currents, we
coexpressed WT and mutant ClC-5 with several rab5 constructs in
Xenopus oocytes. Currents were markedly decreased when WT
ClC-5 was coexpressed with either WT or rab5(Q79L) (Fig.
4A). Conversely, currents were
increased upon expression of rab5(S34N), indicating that constitutively
occurring endocytosis decreases the steady-state level of ClC-5 in the
plasma membrane. Both effects depended on an intact PY motif, because
there was no appreciable effect of any of these rab5 constructs on the
ClC-5(AAE_A) mutant (Fig. 4B).
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We next investigated whether the surface expression and currents of
ClC-5 can be modulated by coexpressing a Nedd4-related protein. We
chose WWP2 because its fourth WW domain interacted strongly with a
synthetic peptide encompassing the PY motif of ClC-5 (18). Like Nedd4,
WWP2 comprises an amino-terminal C2 domain, four WW domains, and a
carboxyl-terminal HECT domain with a predicted ubiquitin-protein ligase
activity. Northern analysis revealed that WWP2 is expressed in all
tissues examined (Fig. 5A).
This includes kidney, suggesting that interaction with ClC-5 might
occur under physiological conditions. Xenopus oocytes have endogenous Nedd4-like activities, sometimes obscuring effects of
overexpressing Nedd4 (27, 28). Therefore several nonfunctional WWP2
constructs were generated that might antagonize the effect of oocyte
homologs (Fig. 5B). In the first construct, the HECT domain
was deleted by truncation (termed WWHECT). The next construct consisted only of WW domains 3 and 4 (termed WW3-4), and the third mutant carried a Cys to Ala point mutation in the HECT domain at a site
that is necessary for ubiquitin binding (termed WWP2CA) (28, 29).
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When WT WWP2 was coexpressed with ClC-5, no effects on the current were
observed. However, coexpression with any of the three nonfunctional
WWP2 constructs led to an approximately 2-fold increase of currents
(Fig. 5C). No such effect was found for the ClC-5(AAE_A) mutant (Fig. 5D). Thus, WWP2 mutants probably antagonize
effects of an endogenous Nedd4-like protein that normally
down-regulates ClC-5 currents. This interaction depends on an intact PY
motif in ClC-5. Furthermore, these experiments strongly suggest that an
intact HECT domain on the WW-containing protein is necessary for the
down-regulation of ClC-5 plasma membrane expression. Additional experiments (data not shown) demonstrated that the increase in currents
elicited by the WWP2 mutants correlated again with an increased surface
expression of ClC-5.
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DISCUSSION |
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ClC-5 is the first chloride channel with a proven role in endocytosis. It is located predominantly in the membranes of vesicles of the (early) endocytotic pathway (7, 8), and its mutational inactivation in man (6) and mice (9) leads to a severe defect in proximal tubular endocytosis. At least upon heterologous expression, a fraction of ClC-5 also resides in the plasma membrane (7, 10). In this work, we have identified an internalization signal in the carboxyl terminus of ClC-5, which, most likely by interacting with ubiquitin-protein ligases, limits its stability in the plasma membrane. This motif and its function closely resembles the PY motif identified in the ENaC (13-17).
The ENaC plays a crucial role in the regulation of distal tubular
sodium reabsorption in the kidney. This process is exquisitely regulated by aldosterone and requires a short half-life of the channel
in the plasma membrane. All three different subunits (,
, and
) carry proline-rich PY motifs in their carboxyl termini. These
motifs are important for the ubiquitination, internalization, and
degradation of the channel. The physiological importance of these
motifs is impressively illustrated by the fact that human mutations
that either delete or mutate PY motifs in Liddle's syndrome (13, 17,
30) lead to hypertension. This is a consequence of enhanced renal
sodium reabsorption resulting from an increased surface expression of
ENaC. However, an increase in open probability was also reported (31).
Using a yeast two-hybrid screen, Staub et al. (14)
identified the ubiquitin-protein ligase Nedd4 as a protein that
interacts with the PY motif via its WW domains. Subsequent work showed
that the regulation of ENaC by Nedd4 involves ubiquitination of the
channel protein (15, 28).
The finding that ClC-5 carries a signal resembling the ENaC PY motif in its carboxyl terminus prompted us to investigate whether it performs a similar role in this largely endosomal chloride channel. Indeed, similar to the findings with ENaC, currents of ClC-5 were increased when the PY motif was mutated. This roughly 2-fold increase in currents correlated with an increased protein level in the plasma membrane, whereas the overall amount of the channel protein was unaffected. This strongly suggested that changes in trafficking, probably in the endocytotic removal from the plasma membrane, underlie the current increase. Indeed, in experiments in which the plasma membrane insertion of newly synthesized channels was prevented by brefeldin A, the apparent half-time of ClC-5 plasma membrane residence was drastically increased by mutations in the PY motif. The half-time of WT ClC-5 in the plasma membrane (20-24 h) was much longer than the 1-3.5 h described for ENaC (15, 16). However, ENaC may be a special case because it needs to be tightly and rapidly regulated in response to changes in salt and fluid balance. Because brefeldin A does not prevent the recycling of endocytosed ClC-5 back into the plasma membrane, the increased half-life of the PY motif mutant at the surface could also be interpreted in terms of an altered trafficking from endosomes to lysosomes. In this model, the mutants would undergo endocytosis at a rate comparable with WT, but the lack of ubiquitin conjugation allows them to evade targeting to the lysosomes. Channels lacking a functional PY motif could then be recycled back to the plasma membrane.
The notion that the PY motif in ClC-5 plays a role in endocytosis is strongly supported by experiments investigating the effects of rab5. Stimulating endocytosis by coexpressed WT or rab5(Q79L) or inhibiting endocytosis by rab5(S34N) decreased or increased ClC-5 currents, respectively. Importantly, this effect was virtually abolished when the PY motif on ClC-5 was inactivated. This suggests that rab5-dependent endocytosis of ClC-5 does not occur via an unspecific retrieval of membranes containing ClC-5 but depends on specific interactions with the PY motif.
Using a homology screen, Pirozzi et al. (18) have previously isolated several cDNAs encoding proteins with multiple WW domains. Glutathione S-transferase fusion proteins containing these domains were tested in vitro for binding to synthetic peptides containing PY-like motifs from several proteins, including ClC-5. The strongest binding to the ClC-5 peptide occurred with the fourth WW domain of a protein dubbed WWP2. WW domains of the other two WWP proteins also displayed some binding. In contrast, at least the first WW domain of rat Nedd4 was unable to bind to a synthetic peptide encompassing the PY motif of ClC-5 (32), showing the specificity of the interaction between WW domains and PY motifs. We have shown here that the most strongly interacting protein, i.e. WWP2, is coexpressed with ClC-5 in kidney. Therefore we chose this protein for our studies. However, we cannot exclude the possibility that ClC-5 shows physiologically more important interactions with other proteins containing WW domains. Similar to Nedd4, WWP2 has an amino-terminal C2 domain that is thought to mediate Ca-dependent lipid binding, four WW domains, and an amino-terminal HECT domain that is predicted to function in ubiquitin conjugation.
Coexpressing the WT form of WWP2 with ClC-5 in Xenopus oocytes had no discernible effects on chloride currents. This may be due to the fact that Xenopus oocytes express a Nedd4 homolog (33) and that its activity on ClC-5 is already near maximal. This contrasts with the recent observation (27, 28) that overexpressing Nedd4 reduced ENaC currents in Xenopus oocytes. The reason for this difference is currently unclear and may reflect e.g. different binding affinities. On the other hand, the stimulation of ClC-5 currents and surface expression by bona fide inactive WWP2 mutants strongly suggest that WW domain proteins interact with the PY motif of ClC-5 and that the mutants act by competing with endogenous Xenopus oocyte homologs. Mutants lacking an HECT domain or carrying a point mutation that interferes with ubiquitin binding (28, 29) increased currents in the presence of an intact PY motif on ClC-5. This strongly suggests that ubiquitination plays a role in the PY motif-dependent endocytosis of ClC-5. Similar experiments also suggested that ENaC is ubiquitinated by Nedd4 (14, 28), and this could also be shown directly (15). Despite many attempts, however, we were unable to show that ClC-5 is ubiquitinated. Ubiquitinated ClC-5 may be short-lived and may therefore have escaped detection, a problem compounded by the fact that many proteins targeted for endocytosis carry only a single ubiquitin moiety (34). Alternatively, ubiquitination may occur on a third protein that might be associated with ClC-5. The unexpected voltage dependence of its currents has previously led to speculations that there may be an additional, as yet unknown, subunit that modifies its voltage dependence (10, 11).
In summary, we have shown that a PY motif in the carboxyl terminus of
ClC-5 modulates its retention in the plasma membrane. Similar to
Nedd4-mediated endocytosis of ENaC, this very likely involves the
interaction with the WW domain of a ubiquitin-protein ligase. Because
ClC-5 is mainly present in endosomal compartments where it is essential
for endocytotic trafficking, its partial presence in the plasma
membrane may be a side effect of recycling. It will be important to
determine whether the interaction of its PY motif with WW
domain-containing proteins is important for its cellular function,
namely its crucial role in both receptor-mediated and fluid-phase
endocytosis (9), and whether this interaction determines sorting
processes further downstream in the endosomal pathway as well.
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ACKNOWLEDGEMENT |
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We thank Willy Günther for constructing the ClC-5(AAE_A) mutant.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to T. J. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Ph.D. student of the faculty of biology, chemistry, and pharmacy
of the Freie Universität Berlin.
§ Present address: Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt am Main, Germany.
¶ To whom correspondence should be addressed. Tel.: 49-40- 4717-4741; Fax: 49-40-4717-4839; E-mail: Jentsch@plexus.uke.uni- hamburg.de.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M010642200
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ABBREVIATIONS |
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The abbreviations used are:
CBS, cystathionine- synthase;
ENaC, amiloride-sensitive epithelial sodium
channel;
HECT, homologous to E6-AP carboxyl terminus;
HA, hemagglutinin;
WT, wild type;
BSA, bovine serum albumin;
BFA, brefeldin
A;
I-V, current-voltage.
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