From the § Program in Membrane Biology & Renal Unit,
Massachusetts General Hospital, and Department of Medicine, Harvard
Medical School, Boston, Massachusetts, 02129-2020, the
Laboratory of Renal Biochemistry, L.C. Simard Research
Center, CHUM & GRTM, Université de Montréal,
Montréal, Québec, H2L4M1 Canada, the ¶ Centre
de Recherche en Rhumatologie et Immunologie, Centre de Recherche du
CHUL, Université Laval, Sainte-Foy, Québec, G1V4G2
Canada, and the
Department of Cell Biology, University of
Virginia Health Sciences Center, Charlottesville, Virginia
22908
Received for publication, December 21, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Kidney proximal tubule epithelial cells have an
extensive apical endocytotic apparatus that is critical for the
reabsorption and degradation of proteins that traverse the glomerular
filtration barrier and that is also involved in the extensive recycling
of functionally important apical plasma membrane transporters. We show
here that an Arf-nucleotide exchange factor, ARNO
(ADP-ribosylation factor nucleotide
site opener) as well as Arf6 and Arf1 small GTPases are
located in the kidney proximal tubule receptor-mediated endocytosis
pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes
is pH-dependent. In proximal tubules in situ,
ARNO and Arf6 partially co-localized with the V-ATPase in apical
endosomes in proximal tubules. Arf1 was localized both at the apical
pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in
purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the
recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal
membranes. The translocation of both ARNO and Arf6 was reversed by
V-type ATPase inhibitors and by uncouplers of endosomal intralumenal
pH, and was correlated with the magnitude of intra-endosomal
acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective
recruitment of ARNO and Arf6 to proximal tubule early endosomes. This
mechanism may play an important role in the pH-dependent
regulation of receptor-mediated endocytosis in proximal tubules
in situ.
In eukaryotic cells, exo- and endocytotic pathways contribute to
various important functions such as secretion and uptake of soluble
proteins as well as recycling of membrane proteins. Receptor-mediated
endocytosis (RME)1 is used by
eukaryotic cells to internalize and process various macromolecules such
as signaling molecules, proteins, nutrients, and toxins. In kidney
epithelial cells, vesicular trafficking plays a crucial role in many
processes, including fluid, ion, and metabolite homeostasis (1). In
particular, kidney proximal tubule epithelial cells are highly
specialized for protein reabsorption and membrane protein recycling via
the RME pathway.
Recently, important progress has been made in our understanding of the
regulation of exo- and endocytotic pathways. Key roles are played by
V-type ATPase (V-ATPase)-dependent intravesicular acidification as well as by the ADP-ribosylation factor (Arf) subfamily
of small GTPases. Earlier studies on the localization and distribution
of overexpressed Arf proteins in cultured cells demonstrated that Arf1
was exclusively associated with the Golgi complex (2, 3) where it is
required for Arf small GTPases have low intrinsic GTPase activity, yet nucleotide
exchange is required for their function as a "molecular switch"
during the GTP/GDP cycle and cytosol to target membrane shuttling.
Regulatory factors including Arf-specific GTPase-activating proteins
(Arf-GAP) and guanine nucleotide exchange factors (Arf-GEF) are,
therefore, essential for Arf function. ARNO
(ADP-ribosylation factor nucleotide
site opener) is a member of a family of Arf-GEFs which also
includes cytohesin-1 (17), GRP1 (18), and EFA6 (19). ARNO is located at
the plasma membrane and in contrast to another Arf-GEF activity located
in the Golgi complex, it is not sensitive to brefeldin A (2, 20).
Recently the insulin receptor-dependent translocation of
ARNO from cytosol to plasma membrane of adipocytes in vivo
was demonstrated (21) and its coimmunoprecipitation with the insulin
receptor has been reported (22). ARNO is 4 times more efficient in
activating Arf6 than Arf1 during exchange of GDP by GTP in
vitro (23), indicating that its physiological function may be to
regulate Arf6 activity.
Numerous mammalian intracellular organelles and carrier vesicles
including clathrin-coated vesicles, endosomes, lysosomes, and the
Golgi/TGN complex have an acidic lumen generated by a V-ATPase in
conjunction with a parallel chloride conductance (24-26). Individual
endocytotic vesicles from kidney proximal tubules showed considerable
pH heterogeneity (27), suggesting that endocytotic trafficking is
associated with a progressive acidification as internalized content
passes from early endosomes to late endosomes and finally to lysosomes.
Two central questions need to be answered. 1) What are the molecular
mechanisms by which this differential acidification in the RME pathway
is achieved? 2) What is the functional role of the intra-endosomal
acidification process in regulation of the RME pathway? One potential
regulatory mechanism involves a pH-dependent interaction of
transport vesicles with Arf small GTPases and other cytosolic coat
proteins. Previously, a pH-dependent interaction of Arf
protein(s) with pancreatic microsomal vesicles (28, 29) as well as
Materials and Antibodies--
Wheat germ agglutinin, aprotinin,
pepstatin A, chymostatin, and phenylmethylsulfonyl fluoride,
concanamycin A (folimycin), FCCP, and nigericin were purchased from
Sigma. Acridine orange was from Molecular Probes (Eugene, OR). ATP,
creatine phosphate, creatine phosphokinase, and bacterial collagenase A
(from Clostridium histolyticum) were obtained from Roche
Molecular Biochemicals (GmbH, Germany). Non-hydrolyzable analogs of the
guanine nucleotides GTP
Production and purification of recombinant rec-Arf1 and rec-Arf6
proteins as well as production and characterization of the rabbit
polyclonal anti-Arf1 (SYL1) and monoclonal anti-Arf6 (SYL6) antibodies
have been previously described (15). The polyclonal anti-ARNO
antibodies (Lap18) were raised against recombinant ARNO. The chicken
polyclonal anti-V-ATPase (subunit E, 31 kDa) antibodies were generated
against a KLH-coupled C terminus peptide (C-GANANRKFLD) and affinity
purified using Pierce "Sulfolink" column (Rockford, IL). They were
generously provided by Dr. Sylvie Breton, MGH Program in Membrane
Biology, and have been characterized previously (34). Rabbit polyclonal
anti-megalin (a proximal tubule apical membrane receptor) antibodies
were obtained from Dr. R. T. McCluskey, Department of Pathology,
Massachusetts General Hospital (35). Mouse monoclonal anti-TGN38 (Clone
2) (trans-Golgi network marker) antibodies were obtained from
Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-Rab11
antibodies (recycling endosome marker) were purchased from
Zymed Laboratories Inc. (San Francisco, CA). Mouse monoclonal anti- Expression, Distribution, and Co-localization of Endogenous ARNO
and Arf Isoforms in Kidney Proximal Tubules in
Situ--
Immunofluorescence experiments were performed on cryostat
sections of rat kidney using an antigen retrieval technique (36). Kidneys from Harlan Sprague-Dawley rats were fixed in
paraformaldehyde-lysine periodate by intravascular perfusion. Kidney
slices were further fixed overnight at 4 °C, before being stored in
phosphate-buffered saline (PBS). Kidney slices were cryo-protected in
30% sucrose/PBS, mounted for cryosectioning in Tissue-Tek embedding
medium and quick-frozen in liquid nitrogen. Sections were cut at a
thickness of 5 µm on a Reichert-Frigocut cryostat and collected on
Fisher Superfrost Plus microscope slides. Antigen retrieval was
performed by treatment of cryostat sections with 1% SDS for 4 min as
previously described (36). After washing in PBS buffer (2 times, 5 min) sections were blocked with 1% bovine serum albumin in PBS for 10 min.
Primary polyclonal anti-ARNO (1:20 dilution), anti-V-ATPase (1:200
dilution), anti-megalin (1:1,000 dilution), anti-Arf1 (1:20 dilution),
anti-Rab11 (1:10 dilution), anti-Rab5 (1:100 dilution), and monoclonal
anti-Arf6 (1:10 dilution) were then applied and incubated overnight at
4 °C. Secondary GAR-Alexa 488 or GAM-Alexa 488 were diluted in DAKO
medium (1:100) and incubated for 60 min at room temperature. After
washing in PBS, slides were counterstained with Evans Blue. In
co-localization experiments, primary mouse monoclonal anti-Arf6, rabbit
polyclonal anti-ARNO, and chicken polyclonal anti-V-ATPase antibodies
were used as above. Primary antibodies were then detected using goat
anti-mouse, goat anti-rabbit, or donkey anti-chicken IgG conjugated to
Alexa 488 or Cy5 as appropriate.
Endocytosis of FITC-dextran by Kidney Proximal Tubules in
Vivo--
FITC-dextran (25 mg in 1 ml of 0.9% NaCl) was injected into
the jugular vein of an anesthetized adult rat. After 10 min kidneys were perfused with PBS followed by paraformaldehyde-lysine
periodate and sucrose solution. After removing the kidneys, they
were immersed in paraformaldehyde-lysine periodate for 2 h for
additional fixation, and FITC-dextran was visualized in 5-µm cryostat
sections as previously described (37).
Confocal and Conventional Immunofluorescence
Microscopy--
Incubated sections were mounted in a 2:1 mixture of
Vectashield mounting medium (Vector Labs, Burlingham, CA) in 1.5 M Tris solution (pH 8.9). Epifluorescence analysis was
performed on a Nikon Eclipse E800 epifluorescence microscope connected
to a Macintosh G4 computer. Images were captured using a Hamamatsu Orca
CCD camera and IPLab Spectrum (Version 3.1a) image processing software
(Scanalytics Inc., Fairfax, VA). Confocal analysis was performed on a
Bio-Rad Radiance 2000 confocal microscope using LaserSharp 2000 software. All images were transferred into Adobe Photoshop 5.0, paginated using Adobe Illustrator 9.0, and printed on a Epson Stylus
Photo750 color printer.
Preparation of Dog Kidney Proximal Tubules in
Suspension--
Cortical tubules (>85% proximal) were prepared from
slices of dog renal cortical tissue. Briefly, both kidneys were removed from anesthetized animals and immediately immersed in ice-cold modified
Krebs-Henseleit saline containing: 120 mM NaCl, 3.2 mM KCl, 1.2 mM KH2PO4,
1.2 mM MgSO4, 0.5 mM
CaCl2, 25 mM NaHCO3, 50 mM mannitol. The renal cortex was sliced with a Stadie
Riggs microtome and tubules were separated and isolated by collagenase digestion as previously described (38, 39). The final suspension of
cortical tubules containing around 60 mg wet weight per ml was kept at
4 °C in Krebs-Henseleit saline fully gassed with 5% CO2, 95% O2 until use.
Purification of Endosomes and Cytosol from Dog Kidney Proximal
Tubules--
Endosomes and cytosol from dog kidney proximal tubules in
suspension were also prepared as previously described (39). Briefly, the suspension of cortical tubules was homogenized in the presence of
protease inhibitors: 0.1 µM aprotinin, 1 µM
pepstatin A, 10 µM chymostatin, and 100 µM
phenylmethylsulfonyl fluoride. The suspension was then centrifuged at
7,700 × g for 15 min and the supernatant was
recentrifuged at 20,000 × g for 30 min. The
supernatant from the second centrifugation was recentrifuged at
150,000 × g for 1 h and was kept on ice until
use. The pellet containing a mixture of brush-border membrane (BBM)
vesicles and early endosomes was collected and homogenized by
aspiration through a 25 5/8-gauge steel needle and recentrifuged at
1,900 × g for 15 min. Endosome-enriched BBM vesicles
were pelleted by centrifugation at 31,000 × g for 30 min, resuspended in 150 mM KCl, 5 mM Tris-Hepes
(pH 7.4) (1 mg of protein/ml), and used to purify the early endosomal
fraction. To separate early endosomes (E) from BBM vesicles (BBMV) a
wheat germ agglutinin negative selection technique was employed as
previously described (39). Protein concentration of proximal tubules,
early endosomal, and 150,000 × g cytosol fractions was
measured after solubilization of membranes in 0.1% SDS with Pierce
bicinchroninic acid protein assay kit (Rockford, IL) using albumin as
standard (40). Freshly prepared endosomes were resuspended to yield
5-10 mg of protein/ml and stored on ice until use. Estimation of the purity of the endosomal preparation (39) as well as characterization of
their acidification machinery (41) was also previously described in
detail. In our experiments, we used only freshly prepared early endosomal and cytosolic fractions to measure endosomal acidification and ARNO/Arf translocation. Freezing in liquid nitrogen with consequent thawing drastically diminished the acidification capacity of endosomes and interfered with the translocation assay (data not shown).
Purification of Golgi Stacks from Kidney Tissue--
Isolation
of Golgi stacks from kidney cortex was performed using a discontinuous
sucrose gradient as previously described (42).
Endosomal Acidification Assay--
Acidification of
endosomes was measured simultaneously with translocation of ARNO/Arf
under the same conditions, and using freshly prepared endosomal and
cytosolic fractions. Endosomal acidification was measured at 37 °C
by acridine orange fluorescence quenching as previously described (41).
Briefly, 100 µg of endosomal protein was added to 2 ml of
acidification/translocation buffer (1.5 mg/ml cytosol supplemented with
150 mM KCl, 50 mM Tris-Hepes (pH 7.4), 5 mM MgSO4, 1 mM ATP, 5 mM creatine phosphate, 10 units/ml creatine phosphokinase,
5 µM acridine orange) to initiate proton transport.
Fluorescence measurements were performed using a Deltascan Model
RFM-2001 spectrofluorimeter (Photon Technology International, South
Brunswick, NJ) with excitation at 450 nm (slit width 1 nm) and emission
at 525 nm (slit width 2 nm). Fluorescence was recorded using the
FelixTM software. Folimycin (0.1 µM) or DCCD
(100 µM) were used to inhibit the V-ATPase while FCCP (1 µM), nigericin (1 µM), or
NH4Cl (10 mM) were used to dissipate
intra-endosomal proton gradients.
In Vitro ARNO, Arf6, and Arf1 Translocation Assay--
The ARNO
and Arf translocation assays were performed in the same buffer used for
the acidification measurements (see above), except that 5 µM acridine orange was replaced with 100 µM phenylmethylsulfonyl fluoride. Also, for the study of
GTP/GDP cycle-dependent Arf translocation, the
ATP-regenerating system (1 mM ATP, 5 mM
creatine phosphate, 10 units/ml creatine phosphokinase) was replaced by
GTP SDS-PAGE and Western Blot Analysis--
Electrophoresis was
performed using 12% SDS-Tris glycine-polyacrylamide gels (SDS-PAGE)
according to Laemmli (43). Endosomal membranes collected after the
in vitro translocation assay were applied to the gel (20 µg of protein per lane) and submitted to electrophoresis using a
conventional Mini-Protean II electrophoresis cell (Bio-Rad). A graphite
electroblotter system MilliBlotTM (Millipore Corp.,
Bedford, MA) was used to transfer proteins from the gels to Immobilon-P
polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA).
Nonspecific binding sites were blocked by exposing the membrane to 5%
nonfat dry milk for 1 h. Membranes were incubated for 1 h
with an antibody against Arf6 (1:1,000 dilution) or Arf1 (1:1,000
dilution) diluted in TBS/Tween/albumin buffer (15 mM NaCl,
0.1% (v/v) Tween 20, 3% (w/v) bovine serum albumin, 5 mM
Tris-HCl, pH 7.0). After washing four times in TBS/Tween buffer, the
membranes were exposed to horseradish peroxidase-conjugated donkey
anti-rabbit or sheep anti-mouse antibodies (dilution 1:3,000) in
TBS/Tween/milk buffer for 1 h. Membranes were then washed four times in TBS/Tween buffer and proteins revealed using the ECL technique. Quantification of Arf6 and/or Arf1 was made using highly purified rec-Arf1 and rec-Arf6 calibration curves running
simultaneously. Quantitative densitometric analysis was performed using
NIH Image 1.62 software.
Two-dimensional PAGE Analysis of Soluble Cytosolic Proteins of
Dog Kidney Proximal Tubules--
Mapping of Arf proteins in the
cytoplasm from dog kidney proximal tubules was performed by
two-dimensional gel electrophoresis (two-dimensional PAGE) and Western
blot analysis. A combination of isoelectric focusing (IEF) (3/10
ampholyte, Bio-Rad) and 12% SDS-PAGE was used to resolve 25 µg of
protein from the 150,000 × g cytosolic fraction of dog
kidney proximal tubules. Electrophoresis was carried out using a
Mini-Protean II 2D (Bio-Rad) electrophoresis system. Electrotransfer of
proteins from the two-dimensional gels to Immobilon-P polyvinylidene
difluoride membranes and immunoblotting with anti-Arf1 (polyclonal
SYL1, 1:1,000 dilution) and anti-Arf6 (monoclonal SYL6, 1:1,000
dilution) antibodies were performed as described above.
Statistical Analysis--
All experiments were performed at
least three times and data were analyzed using appropriate ANOVA
analyses (SuperANOVA software, Abacus).
Endogenous ARNO and Arf6 Are Co-localized with V-ATPase in Apical,
Acidifiable Endosomes in Proximal Tubules--
By confocal
immunofluorescence, endogenous ARNO, an Arf-GEF, was detected in the
apical pole of proximal tubules in association with numerous vesicles
immediately below the apical brush-border membrane and co-localized
with V-ATPase in acidifiable endosomes (Fig.
1A). Small GTPase Arf6 was
also detected in the apical pole of proximal tubules in association
with numerous vesicles and co-localized with V-ATPase in acidifiable
endosomes (Fig. 1B). Moreover, confocal immunofluorescence
analysis revealed that Arf6 co-localized with its exchange factor ARNO
in apical proximal tubule endosomes in situ (Fig.
1C).
The Apical Domain of Proximal Tubules contains Numerous Endocytotic
Vesicles and Corresponds to the Active RME Pathway of These
Cells--
As shown in Fig. 2
(panels A-C), internalized FITC-dextran was concentrated in
endosomal vesicles immediately below the apical brush-border membrane
of kidney proximal tubules 10 min after FITC-dextran injection into the
jugular vein of rats in vivo. The apical receptor protein
megalin (Fig. 2D) showed a similar pattern of apical
localization, immediately below the apical brush-border membrane.
Antibodies against Rab5 (an early endosome marker) and Rab11 (a
recycling endosome marker), also label subapical compartments in
proximal tubule epithelial cells (Fig. 2, E and
F). In contrast, endogenous Arf1 was detected in the
perinuclear region, probably corresponding to the Golgi complex,
similar to previous findings in cultured LLC-PK1 cells
(44). However, Arf1 was also found associated with apical microvilli
and with vesicles at the base of brush-border membrane (not shown).
These results are in agreement with our previous electron microscopic
data (16) showing that both Arf6 and Arf1 are detectable in the
endocytotic pathway of proximal tubule epithelial cells in
situ. Thus, ARNO, Arf6, and V-ATPase (Fig. 1) in situ
are co-localized in apical acidifiable endosomes corresponding to the
active RME pathway of these cells in vivo.
Isolated Kidney Proximal Tubule Endosomes Are Not Contaminated with
Golgi and/or trans-Golgi Network (TGN) Membranes and Contain ARNO,
Arf6, Arf1, and V-ATPase--
We used a previously developed method to
purify endosomes from proximal tubules (39). These endosomes contain
megalin, an apical receptor protein, as well as the endosomal markers
Rab5 and Rab11 (Fig. 3A),
consistent with the apical localization of these proteins in tissue
sections. The purified endosomes were not contaminated with Golgi
(GP58) and/or trans-Golgi network (TGN38) membranes (Fig.
3B) and had a lower amount of V-ATPase-dependent Recruitment of ARNO from Cytosol to
Endosomal Membranes--
Because ARNO was present and co-localized
with V-ATPase in endosomal vesicles in situ (Fig.
1A), we next determined whether the acidification status of
these vesicles was involved in recruiting ARNO to endosomal membranes
in vitro. Purified endosomes were capable of significant
ATP-dependent acidification, reaching a maximum and
steady-state in 10 min (Fig.
4A). This acidification was
dissipated by the V-ATPase inhibitor folimycin, and/or by the
uncoupler, FCCP. Moreover, endosomal acidification was abolished by 0.1 µM folimycin and significantly diminished by 100 µM DCCD (Fig. 4A). The amount of ARNO
associated with purified, non-acidic endosomes was low and varied among
different endosomal preparations (compare Figs. 4, B,
control, and C, control). However, translocation assays
revealed that under conditions of maximal endosomal acidification, a
significant amount of ARNO was recruited from the cytosol to endosomal
membranes (Fig. 4B, +ATP). ATP-dependent ARNO
translocation was completely abolished by folimycin (Fig. 4B, + ATP + folimycin) and by DCCD (Fig. 4B, + ATP + DCCD). The E subunit of the V-ATPase was
used as an internal standard for endosomal protein loading. The amount
of V-ATPase associated with endosomes did not change during
ATP-dependent ARNO recruitment (Fig. 4B). To
demonstrate the specificity of ATP action on ARNO recruitment, we also
examined the effect of a non-hydrolyzable analog of GTP in the ARNO
translocation assay. Incubation of endosomes in the presence of GTP Both Arf6 and Arf1 Are Targeted to Endosomal Membranes during the
GTP/GDP Cycle--
It is generally accepted that Arf1 is recruited
from the cytosol and targeted to Golgi membranes during the GTP/GDP
cycle (2-5). However, the cytosol to membrane shuttling of Arf6 is controversial (9, 33) and target membranes for Arf6 recruitment have
not yet been identified (45). Fig.
5A shows that incubation of
early endosomes with 200 µM GTP V-ATPase-dependent, Selective and Predominant
Recruitment of Arf6 from Cytosol to Endosomal
Membranes--
Acidification-dependent recruitment of Arf
proteins to microsomal membranes has been previously demonstrated (28,
29), but the exact membrane target as well as the precise Arf
isoform(s) involved were not identified. We, therefore, examined the
role of ATP-dependent acidification on the recruitment of
Arf1 and Arf6 to endosomes. A significant amount of Arf6 was recruited from the cytosol to endosomal membranes under conditions of maximal endosomal acidification (Fig. 6A,
+ATP). A significant parallel disappearance of Arf6 from the
cytosol was also observed (not shown). ATP-dependent Arf6
translocation was completely abolished by folimycin (Fig.
6A, + ATP + folimycin) and was
significantly diminished by DCCD (Fig. 6A, + ATP + DCCD). As described above, the amount of V-ATPase
associated with endosomes did not change during acidification (Figs.
4B and 6B). Quantification using rec-Arf6 as a
standard revealed that up to 1.3 µg (per mg of endosomal protein) of
Arf6 was recruited to endosomes under conditions of maximum
acidification (Fig. 6B). In contrast, the
V-ATPase-dependent recruitment of Arf1 from cytosol to
endosomes in our experimental conditions was quite modest compared with
that of Arf6 (Fig. 6A). These data indicate that
acidification-dependent recruitment of Arf6 is specific,
selective, and correlates with recruitment of the Arf-GEF, ARNO (Figs.
4B and 6B).
Translocation of ARNO and Arf6 Correlate with the Level of
Intra-endosomal Acidification--
To further demonstrate the role of
endosomal acidification in ARNO and Arf6 recruitment, experiments with
different uncouplers of endosomal acidification were performed.
Endosomal acidification was completely abolished by 1 µM
FCCP or 1 µM nigericin and was greatly diminished by 10 mM NH4Cl (Fig.
7A). ATP-dependent
Arf6 and ARNO recruitment was prevented or significantly reduced by all
uncouplers tested (Fig. 7B). Both intra-endosomal
acidification and ARNO, Arf6 translocation were reduced in parallel by
different structurally unrelated uncouplers, and the efficiency of
these uncouplers to dissipate the endosomal proton gradient was
correlated with their capacity to prevent translocation of both ARNO
and Arf6 (Fig. 8, A and
B). As shown above, the amount of E subunit of the V-ATPase
associated with endosomal membranes was not affected under these
experimental conditions.
Proximal tubule epithelial cells have an extensive apical
endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier, as well as for the extensive recycling of functionally important apical plasma membrane proteins (1). The physiological importance of acidification processes in proximal tubule function is
underlined by our finding that V-ATPase inhibition in cadmium nephrotoxicity leads to a Fanconi-like syndrome (46) and by our
observation that the V-ATPase inhibitor folimycin completely abolishes
albumin uptake by proximal tubule
cells.2 Moreover, a mutation
in the ClC-5 chloride channel, which together with V-ATPase is believed
to be responsible for efficient endosomal acidification in kidney
proximal tubules, results in Dent's disease, whose manifestations
include a partial Fanconi-like phenotype in humans (47, 48) as well as
in clcn5 Because recruitment of Arf small GTPases to membranes is an early step
in the regulation of both exo- and endocytotic pathways, this process
could be a potential site at which vesicle trafficking and protein
reabsorption in proximal tubules are modulated by pH gradients.
Previous studies have shown that Arf proteins undergo acidification-dependent recruitment to microsomal membranes
(28, 29), but the precise Arf family members that were translocated, the origin of the target vesicles/membranes as well as the molecular mechanism(s) of this recruitment remained unknown. Earlier experiments were performed on mixed microsomal vesicles (containing Golgi, TGN,
endoplasmic reticulum, and endosomal membranes) and Arf proteins were
detected using the monoclonal 1D9 antibody which recognizes all members
of the Arf family except Arf4 (9). Furthermore, while acidification
dependent recruitment of two coat proteins ( Arf-GEF (ARNO) and both Arf6 and Arf1 were detected in the apical RME
pathway in kidney proximal tubules in situ. This
localization pattern partially overlapped with that of megalin, a
proximal tubule apical receptor involved in endocytosis of a broad
range of luminal ligands (33, 35, 49) as well as with the V-ATPase and
an endocytosed marker FITC-dextran. Furthermore, Rab5 a component of
early endosomes and Rab11, a marker of recycling endosomes were also
concentrated in apically located vesicles in these cells. Arf1 was also
found in the perinuclear region (probably the Golgi complex) of
proximal tubule cells in situ. The presence of Arf1 in the
Golgi complex of cultured cells is well documented (2-5, 44). Thus,
these components are poised to participate in the proximal tubule
endocytotic pathway, and this possibility was explored further using
endosomes and cytosol purified from proximal tubules.
Our data show that ARNO and Arf6 are recruited from cytosol to
endosomal membranes in vitro in an
acidification-dependent manner. Recently, recruitment of
ARNO from cytosol to the adipocyte plasma membrane upon activation of
the insulin receptor has been reported (21). Insulin
receptor-dependent recruitment of ARNO to plasma membranes
is dependent on activation of phosphatidylinositol 3-kinase, production
of phosphatidylinositol 3,4,5-P3, and interaction of ARNO
with phospholipid through its C-terminal plekstrin homology domain
(50). However, using specific V-ATPase inhibitors as well as
structurally unrelated uncouplers we found that
ATP-dependent ARNO recruitment is intra-endosomal
pH-dependent. ARNO recruitment was prevented both by
V-ATPase inhibitors (folimycin and DCCD) and endosomal acidification
uncouplers (FCCP, nigericin, and NH4Cl) but not by the
phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 (data
not shown). Also, an effect of endosomal membrane potential on the
recruitment of ARNO could be ruled out since the generation of membrane
potential was minimal under our experimental conditions. All
translocation experiments were performed in the presence of chloride
ions, which due to the presence of as yet unidentified chloride
channels on these endosomal membranes effectively clamp the generation
of membrane potential by the electrogenic V-ATPase, and allows the
development of maximal intra-endosomal acidification (41). Thus, the
ATP effect on ARNO recruitment is most likely to be due to an
acidification-triggered event taking place inside the endosomal lumen
and/or the interior leaflet of the endosomal membrane, and not to a
membrane potential-dependent lipid rearrangement.
This ATP-dependent recruitment of ARNO was accompanied by
the selective, pH-sensitive and predominant recruitment of Arf6 from
cytosol to endosomal membranes, while recruitment of Arf1 was very
modest under the same experimental conditions. A strong correlation
between intra-endosomal lumenal pH and recruitment of both cytosolic
ARNO and Arf6 to endosomes was found. The amount of translocated ARNO
also correlated well with Arf6 recruitment to endosomal membranes. ARNO
is 4 times more effective in activation of Arf6 than Arf1 as a
substrate for GDP/GTP nucleotide exchange (23), providing a potential
explanation for the specificity of the pH effect on Arf6 recruitment.
In contrast to the pH-specific recruitment of Arf6 to endosomal
membranes, both Arf6 and Arf1 were effectively translocated from
cytosol to endosomal membranes by GTP We propose, therefore, that recruitment of ARNO may be an early step in
the pH-dependent translocation of Arf6 from cytosol to
early endosomal membranes for the following reasons: 1) recruitment of
ARNO is dependent only on intra-endosomal acidification but not on the
GTP/GDP cycle; 2) intra-endosomal pH-dependent Arf recruitment is selective and predominant for Arf6; 3)
acidification-dependent translocation of Arf6 is
quantitatively greater than GTP/GDP cycle-dependent Arf6
recruitment; 4) recruitment of Arf6 correlates both with intra-endosomal acidification and ARNO translocation.
Previously, based on the acidification-dependent
recruitment of two coat proteins ( During preparation of this report, Gu and Gruenberg (59) demonstrated
that Arf1 could regulate pH-dependent COP functions in the
early endocytic pathway of BHK-21 cells. GTP We did not address downstream events resulting from the
acidification-dependent recruitment of ARNO and Arfr6 to
endosomal membranes. However, the important role of Arf proteins in the regulation of exocytosis and endocytosis in cultured cells is well
documented (2-11). Arf1, the best studied isoform, is involved in the
recruitment of coat complexes to different organelles of the exocytotic
pathway (60) and to early endosomes (59). Arf1 also regulates binding
of spectrin to Golgi membranes (61) and promotes selective recruitment
of phosphatidylinositol 4-OH(
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP recruitment (4, 5), formation of Golgi-derived
COP-coated vesicles (6), and for formation and maintenance of Golgi
complex structure (7, 8). In contrast, Arf6 was localized to the plasma
membrane of Chinese hamster ovary cells (2, 9). It was proposed
that Arf6 is an important regulator of the RME trafficking pathway (10)
and is also implicated in rearrangement of the actin cytoskeleton (11).
Some of these effects may be mediated via activation of phospholipase D
isoforms by Arf proteins (12, 13). In situ, all six members
of the Arf family have been identified in rat and mouse tissues (14).
Recently we also identified Arf isoforms in human, dog, and rat kidneys
(15, 16).
-COP (30-32) with endosomal vesicles of BHK-21 cells in
vitro was reported. However, the specific members of the Arf
subfamily involved, as well as the molecular mechanism of
pH-dependent recruitment were not established (33). We now report the expression and distribution of ARNO, Arf6, and Arf1 in
kidney proximal tubule epithelial cells and describe their co-localization with apical acidifiable endosomal vesicles in situ. We show that V-ATPase-dependent intra-endosomal
acidification stimulates the recruitment of ARNO and Arf6 from proximal
tubule cytosol to endosomal membranes, implicating this process in
endosomal function in situ.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S and GDP
S were supplied by Calbiochem (La
Jolla, CA). Percoll, low-molecular weight protein calibration kit, IF
standards for two-dimensional PAGE and PhastGel silver staining kit
were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).
Kaleidoscope prestained protein standards and all other reagents for
SDS-PAGE and Western blotting were from Bio-Rad Laboratories.
Immobilon-P transfer polyvinylidene difluoride membranes were obtained
from the Millipore Corporation (Bedford, MO). ECL Western blotting detection kits, donkey horseradish peroxidase-conjugated anti-rabbit antibody, and sheep horseradish peroxidase-conjugated anti-mouse antibody were purchased from Amersham Pharmacia Biotech. Goat anti-rabbit (GAR-Alexa 488) and goat anti-mouse (GAM-Alexa 488) IgG
conjugated to Alexa 488 were obtained from Molecular Probes (Eugene,
OR). Goat anti-rabbit (GAR-Cy5) and goat anti-mouse (GAM-Cy5) IgG
conjugated to Cy5 were obtained from Jackson Immunoresearch (West
Grove, PA).
-COP (Clone M3A5) and anti-GP58 (Clone 58K-9) (marker of the Golgi apparatus) were supplied by Sigma. Rabbit polyclonal anti-Rab5 antibodies (early endosome marker) were obtained from StressGen Corp. (Victoria, BC, Canada).
S (200 µM) and/or GDP
S (200 µM) as
indicated in the figure legends. Freshly prepared endosomes (100 µg)
were mixed with 500 µl of freshly prepared cytosol (1 mg/ml protein)
and incubated for 10 min at 37 °C. Translocation was stopped by
putting the tubes on ice for 2 min, and endosomes were recovered by
centrifugation at 16,000 × g, 4 °C for 60 min. The
endosomal pellet was rinsed with 200 µl of rinsing buffer (200 mM sucrose, 25 mM Hepes-KOH, 50 mM
KCl, 1 mM MgCl2, pH 7, 4). After determination
of protein concentration, endosomes were resuspended in 150 mM KCl, 5 mM Tris-Hepes (pH 7.4), and in
SDS-PAGE sample buffer to give 1 mg/ml protein.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Co-localization of ARNO and Arf6 with
V-ATPase in acidifiable endosomes in situ.
Confocal microscopy analysis of partial co-localization of ARNO with
V-ATPase (A), Arf6 with V-ATPase (B), and ARNO
with Arf6 (C) in kidney proximal tubule apical endosomes
in situ. In panels A and B, primary
anti-ARNO and anti-Arf6 antibodies were detected with GAR-Alexa 488 and
GAM- Alexa 488, respectively (green). Primary
anti-V-ATPase antibodies were detected with DAC-Cy5 (red).
In panel C, primary anti-ARNO antibodies were detected with
GAR-Alexa 488 (green) while anti-Arf6 was detected with
GAM-Cy5 (red). Nuclear (Nuc) staining was
performed with propidium iodide. The right panel in each row
was obtained using the transmitted light mode of the confocal
microscope, and shows the localization of the BBM.
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Fig. 2.
The apical domain of proximal tubules
corresponds to the active RME pathway of epithelial cells and contains
numerous endocytotic vesicles. FITC-dextran (non-proteinaceous
marker for fluid phase endocytosis) is taken by proximal tubules and
concentrated in endosomal vesicles immediately below the apical
brush-border membrane in vivo (A-C). Megalin
(apical receptor for broad range of luminal proteins)(D),
Rab5 (early endosome marker)(E), and Rab11 (recycling
endosome marker)(F) showed a similar pattern of apical
localization, immediately below the apical brush-border membrane.
Tissue sections (D-F) were counter-stained with 1% Evan's
Blue (red fluorescence). Bar = 5 µm.
-COP than the Golgi. Thus,
the presence of Arf6 and particularly Arf1 in the endosomal preparation
together with variable amounts of ARNO and V-ATPase is consistent with
their co-localization in endosomal vesicles in situ (Fig.
3C). Our present and previous kidney fractionation experiments (15, 33) also demonstrate that both Arf6 and Arf1 are
membrane-bound as well as cytosolic proteins. Fig. 3D shows a two-dimensional Western blot analysis of cytoplasmic proteins in the
150,000 × g cytosolic fraction of purified proximal
tubules. The polyclonal anti-Arf1 antibody recognized a single protein (estimated molecular mass
20 kDa and pI
6.0) in
kidney proximal tubule cytoplasm. The monoclonal anti-Arf6 antibody
also recognized a single protein (estimated molecular mass
20 kDa and pI
8.6) in the cytoplasm. There was no
cross-reactivity of these antibodies with other Arf isoforms in kidney
proximal tubules.
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Fig. 3.
Isolated kidney proximal tubule endosomes are
not contaminated with Golgi and/or TGN membranes and contain ARNO,
Arf6, Arf1, and V-ATPase. Purified endosomes are enriched with
megalin receptor and endosomal markers Rab5, Rab11 (A), do
not contain the Golgi/TGN markers GP58, TGN38 (B), and
contain ARNO, V-ATPase, Arf6, and Arf1 (C). Detection and
mapping of Arf6 and Arf1 in the cytoplasm of kidney proximal tubules by
two-dimensional gel electrophoresis and Western blot analysis
(D). The polyclonal anti-Arf1 antibody recognized a single
protein (estimated molecular mass 20 kDa and pI
6.0)
while monoclonal anti-Arf6 antibody recognized a single protein
(estimated molecular mass
20 kDa and pI
8.6) in the
cytoplasm. There was no cross-reactivity of these antibodies with other
Arf isoforms in kidney proximal tubules cytosol.
S
and/or GDP
S did not lead to recruitment of ARNO from the cytosol to endosomal membranes (Fig. 4C).
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Fig. 4.
V-ATPase-dependent translocation
of ARNO from cytosol to endosomal membranes in
vitro. Acidification of endosomes in the presence of
ATP is completely abolished by folimycin and is greatly diminished by
DCCD (A). Acridine orange fluorescence quenching was used to
measure the acidification capacity of freshly prepared endosomes
in vitro. The translocation of ARNO from cytosol to
endosomes is induced in the presence of ATP and this translocation is
abolished by inhibitors of the V-ATPase folimycin and DCCD
(B). Recruitment of ARNO to endosomal membranes is not
affected by GTP S and/or GDP
S (C). The amount of
subunit E of V-ATPase on endosomal membranes does not change during the
experiment and serves as a protein loading control. Molecular sizes
(kDa) of protein markers are indicated. Each in vitro
translocation experiment was performed at least three times.
S but not 200 µM GDP
S led to recruitment of Arf6 from the cytosol to
endosomal membranes. GTP
S-mediated Arf6 recruitment to endosomes was
reduced by GDP
S (lane 4). However, the recruitment of
Arf6 was not completely abolished by GDP
S, probably because the
ratio of GTP
S:GDP
S in the incubation medium was 1:1. A similar
pattern of recruitment during the GTP/GDP cycle was also seen with Arf1
(Fig. 5C). In these experiments, the E subunit of V-ATPase
was also used as an internal standard for protein loading and the
amount associated with endosomes did not change during
GTP-dependent Arf6 and Arf1 recruitment (Fig. 5, A,
B and C, D). Thus, both Arf6 and Arf1 are targeted to
endosomal membranes during the GTP/GDP cycle in vitro. These
results are consistent with the presence of both Arf6 and Arf1 in the
proximal tubule RME pathway in situ.
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Fig. 5.
GTP/GDP cycle dependent recruitment of both
Arf6 and Arf1 from cytosol to endosomal membranes in
vitro. Cytosolic Arf6 is targeted and recruited to
kidney proximal tubule endosomes during the GTP/GDP cycle
(A). Quantification of GTP S-dependent Arf6
translocation using recombinant Arf6 (rec-Arf6) as a standard
(B). Cytosolic Arf1 is also targeted and recruited to kidney
proximal tubule endosomes during the GTP/GDP cycle (C).
Quantification of GTP
S-dependent Arf1 translocation
using recombinant Arf1 (rec-Arf1) as a standard (D). The
amount of subunit E of V-ATPase on endosomal membranes does not change
during the experiment and serves as a protein loading control.
Molecular sized (kDa) of protein markers are indicated. Both cytosol
and endosomes were prepared from purified kidney proximal tubules. Each
in vitro translocation experiment was performed at least
three times.
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Fig. 6.
Selective V-ATPase-dependent
translocation of Arf6 from cytosol to endosomal membranes correlates
with ARNO recruitment. The translocation of Arf6 from cytosol to
endosomes is induced in the presence of ATP and this translocation is
abolished by inhibitors of the V-ATPase folimycin and DCCD
(A). Quantification of selective
V-ATPase-dependent Arf6 translocation and its correlation
with ARNO recruitment (B). Quantification was performed
using recombinant Arf6 (rec-Arf6) and Arf1
(rec-Arf1) as standards. Molecular sizes (kDa) of protein
markers are indicated. Each in vitro translocation
experiment was performed at least three times.
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Fig. 7.
Translocation of ARNO and Arf6 from cytosol
to endosomal membranes is related to the degree of intra-endosomal
acidification. Acidification of endosomes in the presence of ATP
is completely abolished by FCCP and nigericin and is greatly diminished
by NH4Cl (A). Acridine orange fluorescence
quenching was used to measure the acidification capacity of freshly
prepared endosomes in vitro. In the presense of ATP both
ARNO and Arf6 are translocated from cytosol to endosomes and this
recruitment is reduced by various structurally unrelated uncouplers of
intra-endosomal acidification such as FCCP, nigericin, and
NH4Cl (B). The amount of subunit E of V-ATPase
on endosomal membranes does not change during the experiment and serves
as a protein loading control. Molecular sizes (kDa) of protein markers
are indicated. Both cytosol and endosomes were prepared from purified
kidney proximal tubules. Each in vitro translocation
experiment was performed at least three times.
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Fig. 8.
The amount of ARNO and Arf6 recruited from
cytosol to endosomal membranes correlates with the magnitude of
intra-endosomal acidification. A, relative endosomal
acidification and relative ARNO, Arf6 translocation in the presence of
ATP and various intra-endosomal pH uncouplers. B, relative
ARNO and Arf6 translocation from cytosol to endosomal membranes as a
function of relative intra-endosomal acidification. Experimental
conditions were identical to those used in Fig. 7.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
knockout mice (49). However, the link between
endosomal acidification and regulation of the endocytotic pathway in
proximal tubules is poorly understood. Our present data provide new
insight into this process by showing a direct correlation between
V-ATPase-dependent endosomal acidification and recruitment
of the GTP/GDP exchange factor ARNO, and its cognate small GTPase Arf6
to endosomal membranes.
-COP,
-COP) to
endosomes from BHK-21 cells was also reported (30-32), the parallel
translocation of Arf isoforms was not demonstrated in these studies.
S. While both cytosolic (GDP-bound) and membrane (GTP-bound) Arf1 have been found in a variety
of cell types, consistent with its shuttling between the cytosol and
Golgi membranes during the GTP/GDP cycle (4-8), Arf6 was thought to be
an "unconventional" member of the Arf family that was found
exclusively in plasma membranes, but not in endosomes and cytosol, from
Chinese hamster ovary cells (9). Our present data, however, establish
that Arf6 is both a cytosolic and a membrane-bound protein in
situ, and that it cycles from cytosol to endosomal membranes
during the GTP/GDP cycle in a similar manner to Arf1. This is supported
by recent data demonstrating that release of membrane-bound Arf6 into
the cytosol depends upon the presence of physiological concentrations
of magnesium in incubation buffers and that, under appropriate
conditions, Arf6 cytosol-to-membrane shuttling occurs during the
GTP/GDP cycle in variety of cells (45). However, the precise target
membrane for Arf6 was not previously identified (45) and we now show
that early endosomes are a target for both Arf6 and Arf1 recruitment in
kidney proximal tubule epithelial cells.
-COP and
-COP) to endosomes
from cultured BHK-21 cells, the presence of a hypothetical pH-sensitive
protein (PSP) in endosomal membranes was proposed by Gruenberg and
co-workers (30-32). However, neither Arf isoform nor Arf-GEF protein
translocation was reported, and a direct interaction of PSP with
-COP was proposed. To explain our present observations, we
hypothesize that endosomes from kidney proximal tubules also contain a
transmembrane PSP with which ARNO could directly interact upon
endosomal acidification. Arf-GEF proteins including ARNO, cytohesin-1,
GRP1, and EFA6 share common domains that are involved in
protein-protein and protein-lipid interactions as well as GDP/GTP
exchange (17-19, 51). V-ATPase-dependent intra-endosomal
low pH could trigger a conformational change of PSP on an
intra-endosomal site that is transmitted to the cytosolic side of the
endosomal membrane, resulting in signaling and interaction with and
recruitment of ARNO. The existence of such a hypothetical PSP and its
signaling in the endosomal pathway obviously remains to be
demonstrated, but various proteins have been reported to undergo
conformational transitions due to pH changes (52-55). Furthermore, direct interaction of the transmembrane integrin
2-receptor with an Arf-GEF family member (cytohesin-1)
through its Sec7 domain, leading to the recruitment of cytohesin-1
followed by Arf1 from cytosol to plasma membrane, has also been
reported (56). It has also been shown that the association of
intra-Golgi ligands with the KDEL receptor promotes interaction of the
cytoplasmic tail of this receptor with an Arf1-specific GAP
(Arf1-GAP1), providing another example of modulation of the Arf pathway
by putative conformational changes in a transmembrane protein (57,
58).
S had a very small, if
any, effect on translocation of Arf6 to endosomal membranes. Recruitment of Arf1, however, was significantly stimulated during the
GTP/GDP cycle and correlated with recruitment of
COP but not
COP onto endosomal membranes. GTP
S-dependent
recruitment of Arf1 was partially (50%) diminished by preincubation of
endosomes with a high concentration (50 µM) of nigericin.
However, a direct effect of ATP and specific V-ATPase inhibitors
(without GTP
S and/or nigericin) on the translocation of Arf isoforms
to BHK-21 cell endosomes was not shown (59). Furthermore, a correlation between endosomal acidification and Arf1 translocation was not demonstrated. In contrast, we show that both endogenous Arf1 and Arf6
(but not ARNO) are recruited from cytosol to endosomal membrane during
the GTP/GDP cycle. These data suggest that Arf1 could play a GTP/GDP
cycle-dependent regulatory role in the apical endocytotic pathway of kidney proximal tubules, perhaps at an early step in endocytotic vesicle budding from the apical membrane, prior to vesicle
acidification. In our experiments both endosomes and cytosol were
purified from the same source (kidney proximal tubules), they were not
concentrated (fractionated) and only endogenous proteins were used. In
this way, we show that endogenous ARNO and Arf6 are specifically and
selectively recruited from cytosol to purified endosomal membranes in a
pH-dependent manner: translocation is promoted by ATP
(without addition of GTP
S but in the presence of endogenous GTP/GDP)
and is reversed either by specific V-ATPase inhibitors or by various
structurally unrelated uncouplers of endosomal acidification. Moreover,
translocation experiments were performed simultaneously and under
identical conditions with acidification assays. We hypothesize that the
first protein to be translocated in an
acidification-dependent (GTP/GDP-independent) manner is ARNO, which triggers the selective GDP/GTP exchange and recruitment of
Arf6. Thus our data suggest that in endosomes of proximal tubule epithelial cells, the hypothetical "pH sensitive protein"
previously proposed by Gruenberg and co-workers (30-32) initially
interacts with ARNO and/or with Arf6.
) and phosphatidylinositol 5-OH kinases
(62). Arf1 directly interacts with
COP (63) and regulates the
activity of phospholipase D1 (12) and phospholipase D2 (13).
Overexpressed Arf6 plays a role in regulating the receptor-mediated
endocytotic pathway of cultured Chinese hamster ovary (10, 64) and
Madin-Darby canine kidney (65) cells as well as the secretory pathway
of bovine adrenal chromaffin cells (66, 67). Arf6 has been also implicated in the regulation of actin cytoskeleton rearrangements (11)
as well as in the regulation of phospholipase D activity. Any or all of
these activities could ultimately be important in the regulation of the
RME pathway by pH-dependent and/or GTP/GDP cycle-dependent
Arf recruitment to proximal tubule endosomes. Diminished recruitment of
ARNO and Arf6 to endosomes with deficient acidification, for example,
during cadmium intoxication (46) and Dent's disease (47-49) might be
a crucial molecular event leading to development of Fanconi syndrome as
depicted in Fig. 9. Thus we propose that
intra-endosomal pH-sensitive recruitment of ARNO and Arf6 to kidney
proximal tubule endosomes could be one possible missing link between
impaired endosomal acidification and defective protein reabsorption in
Dent's disease and Fanconi syndrome. While these and previous data
reveal a relationship between transmembrane signal transduction and
vesicular trafficking, the precise functional roles of ARNO, Arf6, and
Arf1 and their downstream effectors in the RME pathway of kidney
proximal tubules in situ remain to be determined.
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Fig. 9.
Schematic representation of protein
reabsorption through the apical endocytotic pathway in kidney proximal
tubules and the potential role of endosomal acidification in the
physiological regulation of this process by ARNO and Arf6
recruitment. Intra-endosomal pH-sensitive recruitment of ARNO and
Arf6 to kidney proximal tubule endosomes as a possible link between
impaired endosomal acidification and protein reabsorption in Dent's
disease and Fanconi syndrome. A, illustrates efficient
protein reabsorption under normal conditions. ARNO and Arf6
recruitment, driven by the acidic endosomal pH signaling, is required
for sorting in endosomes, and for more distal processes including
trafficking of some internalized proteins to lysosomes for degradation.
The glomerulus/proximal tubule shown in panels A and
B is a photomontage taken from tissue immunostained for Arf6
(arrowheads). LMW proteins, low molecular weight
proteins; AA, amino acids. 1, blood vessel;
2, glomerulus; 3, S1 segment of proximal tubule).
B, illustrates defective protein reabsorption resulting from
deficient endosomal acidification due to inhibition of the V-ATPase
and/or chloride channels. Apical protein reabsorption is blocked due to
deficient ARNO and Arf6 recruitment upon diminishing endosomal
acidification signaling. Reduced apical protein reabsorption leads to
increased protein delivery to more distal segments of the urinary
tubule and to proteinuria, which occurs in Dent's disease and Fanconi
syndrome.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Sylvie Breton for providing chicken polyclonal anti-V-ATPase antibodies used in co-localization experiments. We also thank Mary McKee for technical support with the in vivo FITC-dextran uptake experiments.
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FOOTNOTES |
---|
* This work was supported by Medical Research Council of Canada Grant MT-7875 (to V. M. and P. V.) and National Institutes of Health Grants DK42956 (to D. B.) and DK38452 (to D. A. A., V. M., J. E. C., and D. B.).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.
** To whom correspondence should be addressed: Program in Membrane Biology & Renal Unit, Harvard Medical School, Massachusetts General Hospital East, 149 13th St., Boston, MA 02129-2020. Tel.: 617-724-9815; Fax: 617-726-5669; E-mail: vmarshansky@partners.org.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011577200
2 V. Marshansky, D. A. Ausiello, and D. Brown, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
RME, receptor-mediated endocytosis;
Arf, ADP-ribosylation factor;
GEF, guanine nucleotide exchange factor;
ARNO ADP-ribosylation factor
nucleotide site opener, BHK, baby hamster kidney;
FCCP, carbonyl
cyanide p-trifluoromethoxyphenylhydrazone;
GTPS, guanosine-5'-O-(3-thiotriphosphate);
GDP
S, guanosine-5'-O-(2-thiodiphosphate);
PAGE, polyacrylamide gel
electrophoresis;
PBS, phosphate-buffered saline;
FITC, fluorescein
isothiocyanate;
BBM, brush-border membrane;
DCCD, dicyclohexylcarbodiimide;
TGN, trans-Golgi network;
PSP, pH-sensitive
protein.
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