1 Graduate School of Pharmaceutical Sciences, Kyushu University 3-1-1, Maidashi,
Higashi-ku, Fukuoka, 812-8582, Japan
2 Department of Cell Genetics, National Institute of Genetics, Yata 1111,
Mishima, Shizuoka, 411-8540, Japan
3 Biological Program, Yamanashi Medical University, Yamanashi 409-3898,
Japan
* Author for correspondence (e-mail: himeno{at}phar.kyushu-u.ac.jp)
Accepted 10 October 2002
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Summary |
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Key words: Lysosomes, SKD1, Membrane traffic, Hybrid organelle, Class E vps
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Introduction |
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The family of AAA (ATPases associated with cellular activities)-type
ATPases has been found to play a crucial role in membrane transport. One such
AAA-ATPase is Sec18p/NSF (N-ethylmaleimide-sensitive fusion protein).
NSF is one of the most studied AAA family proteins and appears to work as a
master for either the assembly or dissociation of the SNARE complex at several
steps in the membrane transport process
(Hay and Scheller, 1997).
Another important AAA molecule, Vps4p/End13p, was found in yeast vacuolar
protein sorting (vps) mutants and has been extensively characterized. Vps4p is
a yeast protein known to be required for the efficient transport of newly
synthesized carboxy-peptidase Y (CPY) molecules from the TGN to the vacuole,
which is the yeast counterpart of mammalian lysosomes
(Babst et al., 1997
).
END13 is allelic to VPS4 and mutations in this gene result
in deficiencies in both the endocytic and biosynthetic pathways
(Zahn et al., 2001
) that
result in a phenotype known as class E
(Raymond et al., 1992
). The
most characteristic feature of class E mutants is the appearance of a novel
prevacuolar-like structure adjacent to vacuoles, the so-called class E
compartment. This class of mutants indicates the accumulation of soluble
vacuolar hydrolases, certain Golgi resident proteins and an endocytosed
membrane that can be monitored using the fluorescent lipid dye, FM4-46.
Therefore, Vps4p/End13p is thought to function in late endosomes/prevacuolar
compartments by binding and releasing other class E Vps proteins (Vps24p and
Vps32p/Snf7p) in a nucleotide-dependent cycle
(Babst et al., 1998
). End13p is
also implicated in the formation of internal vesicles present in the
multivesicular body (MVB) (Odorizzi et
al., 1998
).
The mouse homologue of Vps4p/End13p is the
suppressor-of-potassium-transport-growth-defect-1 protein (SKD1)
(Perier et al., 1994). That
SKD1 is involved in crucial membrane transport events is indicated by recent
studies (Bishop and Woodman,
2000
; Nara et al.,
2002
; Yoshimori et al.,
2000
). Expression of a mutant SKD1 molecule, denoted as
SKD1(E235Q), that lacks ATPase activity in mammalian cells, exhibited dominant
negative effects on various membrane transport processes and the formation of
autolysosomes. These cells developed abnormal membranous bodies with some
diversity that contained endocytosed fluid and receptors as well as lysosomal
markers, suggesting they are a mixture of compartments bearing either
endosomal or lysosomal characteristics. Despite the works that characterized
these abnormal compartments, the functional consequences of SKD1(E235Q)
expression on the endocytic and biosynthetic pathways are still not clear. We
show here that the alterations in membrane transport caused by the expression
of SKD1(E235Q) closely resemble features of the class E phenotype in
Saccharomyces cerevisiae. Our observations led us to propose that
SKD1 is a master molecule that drives vesicle formation and/or membrane
fission and thus functions at multiple steps in membrane transport
process.
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Materials and Methods |
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Cell culture
Madin-Darby canine kidney (MDCK), A431 human epidermoid carcinoma, normal
rat kidney (NRK), and rat fibroblast (3Y1-B) cells were cultured in Dulbecco's
modified Eagle's medium (Sigma, St Louis, MO) with 10% fetal bovine serum
(Life Technologies, Gaithersburg, MD). The cultured hepatocyte cell line WIF-B
was grown in F-12 COON's modified medium (Sigma) supplemented with 10 µM
hypoxanthine, 0.04 µM aminopterin, and 1.6 µM thymidine (HAT; Life
Technologies), 50 µg/ml of streptomycin, 200 units/ml of penicillin and 0.5
µg/ml of fungizone (Life Technologies) and 5% fetal bovine serum (Life
Technologies) as described elsewhere
(Ihrke et al., 1993;
Shanks et al., 1994
).
Adenoviral infection
The adenovirus encoding wild-type LDLR was obtained from Enrique
Rodriguez-Boulan (Cornell University, NY). The preparation of adenoviruses
encoding both Cre recombinase and GFP-SKD1(E235Q) has been described elsewhere
(Nara et al., 2002). The cells
were infected in the appropriate culture medium and incubated for 24 hours
prior to conducting the experiments.
Surface binding and internalization of
125I-antibodies
MAb to endolyn (HA501) and control mouse IgG were iodinated with
125I-Na (PerkinElmer Life Sciences, Boston, MA) by using iodogen
(Pierce Chemical Co., Rockford, IL), according to the manufacturer's
instructions. Labeled proteins were separated from free 125I-Na on
a PD10 column (Pharmacia Biotech). NRK cells (approximately
4x104 cells/well) were incubated with iodinated antibodies
(20 µg/ml in 0.5% BSA/DMEM, 20 mM Hepes pH 7.4 containing 40 µM
leupeptin and pepstatin) either for 1 hour at 4°C (surface labeling) or
37°C (internalization) for the indicated times. Subsequently, cells were
washed three times with 0.5% BSA/PBS and PBS, respectively, then solublized
with the lysis buffer (0.5% Triton X-100, 0.25% deoxycholic acid, 25 mM
Tris-HCl pH 7.5). The amount of cell-associated 125I-mAb and
protein concentration in each sample were determined using a -counter
and BCA protein assay kit (Pierce), respectively. To determine the nonspecific
antibody binding or uptake, cells were identically treated with iodinated
control mouse IgG, and these values were normalized with the labeling
efficiency of both antibodies and subtracted from those of HA501. The amount
of surface appearance and internalization of endolyn were represented as the
count (cpm)/µg total cell protein.
Immunofluorescence and antibody transport assay
Cells were fixed with 4% paraformaldehyde for 5 minutes on ice and
permeabilized with methanol for 10 minutes. For the LBPA staining, cells were
fixed with 4% paraformaldehyde for 5 minutes on ice and permeabilized with
0.05% saponin. Fixed cells were rehydrated and washed in PBS for 15 minutes.
After blocking in 1% bovine serum albumin (BSA) in PBS, the cells were
incubated with primary antibodies in 1% BSA/PBS for 1 hour at the following
dilutions: anti-LDLR (mouse ascites; 1:200), anti-ASGPR (rabbit pAb; 1:100),
anti-TGN38 (mouse mAb; 1 µg/ml), anti-endolyn (mouse ascites; 1:200),
anti-human EGFR (mouse mAb; 1 µg/ml), anti-rat cathepsin L (rabbit pAb,
affinity purified; 10 µg/ml), anti-rat lgp120 (mouse ascites; 1:200), and
human lamp1 (mouse hybridoma culture sup; 1:200). The secondary goat
anti-rabbit or anti-mouse antibodies that had been conjugated with Alexa-594,
Cy3 or Cy5 were used at 5-10 µg/ml.
To label lysosomes, cells were preloaded for 1 hour with the appropriate medium containing 40 µg/ml of TR-dex and then chased with normal medium for 12 hours prior to adenoviral transfection. For the antibody transport assay, cells on coverslips were washed once with Hepes-buffered serum-free medium (HSFM), cooled to 4°C and then labeled with primary antibodies in 0.2% BSA/HSFM for 15 minutes. The anti-TGN38 mAb was used at 20 µg/ml and anti-endolyn mAb (HA502; ascites) was diluted 1:100 for surface labeling of the cells. The surface-labeled cells were then transferred, either directly (for TGN38) or after washing three times with ice-cold 0.2% BSA/HSFM (for endolyn), to prewarmed normal medium and incubated for 1 hour at 37°C. The internalized antibodies were detected with either Cy3- or Alexa 594-conjugated goat anti-mouse secondary antibodies (10 µg/ml) and visualized by indirect immunofluorescence.
Immunoelectron microscopy
Adenovirus-infected 3Y1-B cells were fixed at room temperature for 1 hour
with 4% paraformaldehyde and 0.25% glutaraldehyde in 0.2 M Hepes-KOH buffer
(pH 7.4), and washed with PBS. The fixed cells were briefly stained with
0.001% toluidine blue for 2 minutes, detached from culture dishes with a
rubber policeman in the presence of 20% ethanol, and collected by
centrifugation. Cell pellets were then suspended in 1% low-melting temperature
agarose and centrifuged. The cells in pellets were chilled to facilitate
binding to each other. The pellets were cut into small blocks, dehydrated in a
graded ethanol series at -20°C, and embedded in LR White (Polyscience,
Warrington, PA). Thin sections were cut using a diamond knife in a Reichert
Ultracut R and mounted on nickel grids. Sections were immunostained with a
combination of the primary antibodies with protein A-gold probes, and further
stained with uranyl acetate and lead citrate. Sections were then examined with
a Hitachi H7500 electron microscope at an acceleration voltage of 75 kV.
Imaging
All the immunofluorescence images, except for those with triple labeling,
were observed on a Leica DMRB microscope (Wetzlar, Germany) and acquired
through a cooled CCD camera, MicroMAX (Princeton Instruments, Trenton, NJ) and
digitally processed using IPlab Software (Scanalytics, Fairfax, VA). Images of
triple-labeled cells that had been stained with GFP-SKD1(E235Q), TR-dex and
Cy5-labeled anti-mouse antibody were acquired by a confocal microsopy system,
LSM 5 PUSCAL, Carl Zeiss (Thornwood, NY). All images from immunofluorescence
microscopy, western blotting and pulse-chase experiments were assembled and
labeled using Adobe PhotoShop (Adobe Systems, Mountain View, CA).
EGF stimulation and EGFR degradation assays
Both adenovirus-infected and uninfected A431 cells were grown in a 3.5 cm
dish to 80% confluency and incubated for 16 hours with DMEM containing 1%
BSA, after which they were incubated with 500 ng/ml of hEGF for a given
period. The cells were harvested with a 10 mM Tris-HCl (pH 7.4) 0.15 M NaCl
buffer and the protein concentration of the lysate was determined by Lowry's
method (Lowry et al., 1951
).
Total cell lysate (10 µg) of each sample was subjected to SDS-PAGE
according to Laemmli's method (Laemmli,
1970
) using 7.5% acrylamide under reducing conditions, after which
the gel was processed for western blotting according to standard procedures
using enhance chemiluminescence (ECL) detection kit (Amersham Pharmacia). The
remaining EGFR was detected with anti-hEGFR antibody and ECL bands were
quantitated by NIH-image.
Pulse chase and immunoprecipitation
Both adenovirus-infected and uninfected NRK cells were grown in a 3.5 cm
dish to 80% confluency, after which they were metabolically labeled for
15 minutes with 100 µCi/ml [35S]methionine/cysteine
(EXPRESSTM Protein Labeling Mix, [35S]-Easy TagTM, New
England Nuclear, Boston, MA) and chased with normal medium for specific
periods. The labeled cells were lysed with lysis buffer (10 mM Tris-HCl (pH
7.4), 0.15 M NaCl, 0.1% Triton X-100, 1 mM EDTA and protease inhibitor
cocktail (Sigma) and centrifuged in a microfuge for 10 minutes to remove
insoluble compounds. The cell extracts and chasing medium were subsequently
processed for immunoprecipitation with anti-rat cathepsin D antibody and
protein A-agarose (Roche, Indianapolis, IN) as described previously
(Tanaka et al., 2000
). The
immunoprecipitates were analyzed by SDS-PAGE using 10% acrylamide under
reducing conditions. Radioactive bands were detected and quantitated with a
Fuji BAS 1000 Imaging Analyzer.
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Results |
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Previous study has shown that the transient expression of SKD1(E235Q) in
cultured cells led to aberrant endosomes that were highly vacuolated and
mostly positive for SKD1(E235Q) (Bishop and
Woodman, 2000; Yoshimori et
al., 2000
). We found membranous structures that were unusually
enlarged and sometimes swollen but lacked SKD1(E235Q) (Figs
1,
6). We denoted both types of
compartments as `E235Q compartments', because they had been induced only by
expression of SKD1(E235Q).
|
|
Basolateral recycling receptors, LDLR and ASGPR, are trapped in E235Q
compartments
We have examined the possible involvement of SKD1 in the recycling of the
receptors from early and recycling/sorting endosomes to the PM. To examine
whether SKD1(E235Q) expression indeed affects the distribution of LDLR,
Madin-Darby canine kidney (MDCK) cells were co-infected with two adenoviruses,
one expressing LDLR and the other expressing GFP-SKD1(E235Q) and their
intracellular distribution was analyzed by immunofluorescence microscopy. In
the polarized MDCK cells infected with LDLR-expressing adenovirus alone,
virally expressed LDLR localized to the basolateral PM and early endosomes
that were closely aligned to the lateral membrane
(Fig. 1A,B; arrows). The
doubly-infected cells had phase lucent-swollen vacuoles
(Fig. 1C; asterisks) and showed
a significant redistribution of LDLR from PM to the swollen vacuoles, as well
as small vesicles clustered in perinuclear regions, which probably have the
E235Q compartments (Fig. 1A,B;
arrowheads), whereas the extent to which LDLR and GFP-SKD1(E235Q) colocalized
varied between cells. Notably, the cell surface and peripheral endosomal
distribution of LDLR was significantly decreased in SKD1(E235Q)-expressing
cells (Fig. 1B; asterisks).
The asialogylcoprotein receptor (ASGPR) is a hepatocyte-specific receptor that is localized to perinuclear recycling endosomes as well as to early endosomes very close to the basolateral PM in polarized cultured WIF-B hepatocytes (Fig. 1D,E; arrows). Expression of SKD1(E235Q) in WIF-B cells led to the appearance of swollen vacuoles (Fig. 1F; asterisks) as in MDCK cells, but did not alter the cell polarity as judged by the normal apical PM distribution of 5'-nucleotidase (data not shown). The ASGPR in the SKD1(E235Q)-expressing WIF-B cells was redistributed into E235Q compartments. In contrast, there were few or no ASGPR-positive peripheral endosomes near the PM (Fig. 1D,E; arrowheads) in the infected cells. While some ASGPR colocalized with GFP-SKD1(E235Q), the extent of colocalization varied between the cells, similar to what was observed for LDLR (see above). It is likely that the distribution of ASGPR is affected by SKD1(E235Q) expression in a fashion similar to that of LDLR. Thus we conclude that the recycling of receptors from early and recycling endosomes to basolateral PM in polarized epithelial cells was regulated by SKD1 ATPase activity.
Recycling of TGN38 is abrogated by the expression of SKD1(E235Q)
TGN38 and a furin, which localize in TGN, are known to be recycled between
the TGN and the PM via early endosomes
(Ghosh et al., 1998;
Mallet and Maxfield, 1999
;
Reaves et al., 1993
;
Takahashi et al., 1995
;
Voorhees et al., 1995
). TGN38
in SKD1(E235Q)-expressing NRK cells, like LDLR and ASGPR, localized to the
E235Q compartments. Although the majority of TGN38 antibody staining still
represented typical TGN staining as seen in uninfected cells, there was
significant redistribution of TGN38 into a punctate structure near the Golgi
and sometimes into the swollen structures in SKD1(E235Q)-expressing NRK cells
(arrows in Fig. 2A-C).
|
To further test the possibility that the TGN38 in the E235Q compartments is accumulated via endocytic routes, we followed the fate of exogenously applied TGN38-specific antibodies in SKD1(E235Q)-expressing NRK cells. After 1 hour of chase, the internalized TGN38-specific mAb had reached the perinuclear TGN area in uninfected cells (see arrowheads in Fig. 2D,E) whereas, in the infected cells, the mAb accumulated significantly in the E235Q compartments (see arrows in Fig. 2D-F) and could not reach the TGN area. This was not due to the destruction of the TGN structure by overexpression of SKD1(E235Q), because there was no difference in the perinuclear tubular and punctate distribution of AP-1 between the infected cells (asterisks in Fig. 2G,H) and uninfected cells. These observations indicate that, in addition to the receptors recycling between PM and early endosomes, the recycling pathway used by TGN38 is also significantly affected by the endosomal dysfunction caused by the expression of SKD1(E235Q).
SKD1(E235Q) alters the intracellular distribution and the cell
surface appearance of the endosomal-lysosomal membrane protein endolyn
It is known that some lysosomal membrane proteins continuously recycle
between the PM and the lysosomes via early and late endosomes
(Lippincott-Schwartz and Fambrough,
1987). Endolyn is one of lysosomal membrane proteins and belongs
to the highly O-glycosylated mucin-like protein family
(Croze et al., 1989
;
Ihrke et al., 2000
;
Chan et al., 2001
). It was
shown that in polarized hepatic WIF-B cells, a fraction of endolyn
internalized from the basolateral surface is delivered to endosomes/lysosomes
either directly or indirectly via a subapical compartment
(Ihrke et al., 1998
). Recent
work demonstrated that in polarized MDCK cells newly synthesized endolyn is
directed to the apical surface before it reaches lysosomes
(Ihrke et al., 2001
). Indeed,
it has been demonstrated that endolyn contains multiple targeting signals; one
is an N-glycan-dependent apical targeting signals in the luminal
domain of endolyn and the other is basolateral/lysosomal sorting information
contained in the cytoplasmic tail of it
(Ihrke et al., 2001
). To
clarify the effect of SKD1(E235Q) on the recycling pathway between the PM and
lysosomes, we examined the distribution and recycling of endolyn in
SKD1(E235Q)-expressing NRK cells. We found that the distribution of endolyn
was altered by expression of SKD1(E235Q) and the protein was accumulated in
E235Q compartments (Fig. 3A,B,
infected cells are marked with asterisks). Then we tested the effect of
SKD1(E235Q) expression on the endocytic pathway from the PM to the lysosomes
by following the fate of exogenously applied endolyn-specific antibodies in
SKD1(E235Q)-expressing NRK cells. We found that the surface labeling of the
endolyn-specific mAb at 4°C, which indicates the degree of surface
appearance of endolyn, was significantly less on SKD1(E235Q)-expressing cells
than uninfected cells (Fig.
3C,D). The measurement of a cell surface binding of
125I-mAb to endolyn (HA501) further revealed that it was reduced to
approximately 56% of control cells (Fig.
3G). Since about 30% of internalized antibody to endolyn could be
recycled, these results suggest that SKD1(E235Q) leads to the accumulation of
the internalized endolyn to the E235Q compartments and inhibits endolyn
recycling to the PM. This was confirmed by the following results. First, after
a subsequent chase for 1 hour at 37°C, the internalized mAb to endolyn was
localized to the E235Q compartments in the infected cells, while in the
uninfected cells, it was delivered to late endosomes and lysosomes
(Fig. 3E,F). Second, an
internalization of 125I-mAb in the cells expressing SKD1(E235Q) was
reduced to less than 70% of control cells after a 2 hour incubation
(Fig. 3H). Thus, we conclude
that the expression of SKD1(E235Q) caused the perturbation of recycling of
endolyn from endosomes to the cell surface.
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Processing of a newly synthesized lysosomal acid hydrolase, cathepsin
D, into the mature form is impaired by the expression of SKD1(E235Q)
Vps4-deletion and temperature-sensitive Vps4 mutants in yeast
exhibit an accumulation of CPY, a soluble vacuole enzyme in class E
compartments, which delays their maturation from the p2-form to the mature
form and causes the partial secretion of the p2-form
(Babst et al., 1997;
Zahn et al., 2001
). We
examined the effect of SKD1(E235Q) expression on the transport of a newly
synthesized soluble lysosomal enzyme, cathepsin D, to lysosomes by pulse-chase
experiments of NRK cells with [35S]methionine/cysteine. As shown in
Fig. 5, in NRK cells, cathepsin
D was synthesized as a 45 kDa pro-form (pro-catD), and was subsequently
processed to a 43 kDa mature form (mature catD). Although in uninfected cells
proteolytic processing of pro-catD to the mature form was completed after a
chase of 3 hours, in SKD1(E235Q)-expressing cells more than 50% of the newly
synthesized catD remained as a pro-form even after a chase of 3 hours. In
addition to the retardation of proteolytic processing, the expression of
SKD1(E235Q) also resulted in a hyper-secretion of pro-catD into the medium
(Fig. 5, see EQ infection,
right). As in the case of CPY-processing in class E mutants in yeast,
SKD1(E235Q) led to a delay in the processing and hypersecretion of
pro-catD.
|
Expression of SKD1(E235Q) induces the accumulation of hybrid
compartments that bear features of both lysosomes and late endosomes
To analyze the effect of SKD1(E235Q) expression on the morphology of
lysosomes in NRK cells, Texas-Red dextran was preloaded into cells and then
adenovirus was used to infect cells. In uninfected cells, TR-dex-labeled
lysosomes can be observed as fine punctates that are mostly found near the
nucleus but are also present throughout the cell
(Fig. 6A). However, in infected
cells (Fig. 6A, see asterisks)
the TR-dexlabeled lysosomes were significantly less frequent (average
number/cell is 10.6±4.2 versus 58.4±21.4 in normal cells) and
were not concentrated near the nucleus but scattered throughout the cells. The
lysosomes in the infected cells were also considerably larger than those in
uninfected cells (average diameter is 1.19±0.47 µm versus
0.48±0.26 µm, range is 0.8-2.0 µm versus 0.2-0.8 µm)
(Fig. 6E). Interestingly, these
enlarged TR-dex-labeled structures were mostly not associated with
GFP-SKD1(E235Q). The enlarged lysosomes were also positive for cathepsin L and
lysobisphosphatidic acid (LBPA) (Kobayashi
et al., 1998), respectively
(Fig. 6B,C). Together these
observations suggest that the enlarged lysosomes induced by the expression of
GFP-SKD1(E235Q) have characteristics of both late endosomes and lysosomes and
thus they may be hybrids of these two organelles. Triple labeling of the cells
by infection with the GFP-SKD1(E235Q)-expressing adenovirus, pre-loading with
TR-dex and staining with anti-lgp120 antibody revealed the existence of two
distinct lgp120-positive compartments in the infected cells
(Fig. 6D). In uninfected cells,
all TR-dex positive compartments perfectly colocalized with lgp120 (see purple
markings in Fig. 6D), which
indicates that these compartments are lysosomes. In the infected cells (see
asterisk in Fig. 6D), the
enlarged hybrid organelles (TR-dex+, lgp120+) poorly colocalized with
GFP-SKD1(E235Q) (see arrowheads in Fig.
6D). This notion may imply that the accumulation of hybrid
organelles is not due to a direct effect of SKD1(E235Q) but an indirect one,
such as a depletion of the ADP-bound form of SKD1 from the cytosol. However,
in these cells, some lgp120 signals were also observed in the
GFP-SKD1(E235Q)-positive compartments (see light blue markings in
Fig. 6D) that lack TR-dex (see
arrows in Fig. 6D). In
addition, there are many structures that are only labeled with
GFP-SKD1(E235Q), and that appear to be aberrant and large vacuole-like
structures. These latter structures may be derived from the direct association
of SKD1(E235Q) with their membrane and correspond to the accumulation of early
endosomes and/or intermediate compartments between early and late
endosomes.
The immunofluorescence data were supported by immunoelectron microscopy. In control cells (Fig. 7A,B), both electron dense lysosomes (arrows) and MVB-like late endosomes (arrowheads) were labeled with an anti-LGP107 pAb and their diameter was less than 1 µm. In contrast, in GFP-SKD1(E235Q)-expressing cells (Fig. 7C-F), there were many aberrant membranous structures, which were positive for LGP107. We found that MVBs tended to cluster near the nucleus, docked and fused with each other (arrows in Fig. 7C,E,F). In addition, the typical hybrid organelle-like structures, MVBs fused with electron dense lysosomes, were frequently observed (arrowheads in Fig. 7C,E). Interestingly, consistent with the immunofluorescence analysis, the diameter of the MVBs was over 1 µm (asterisks in Fig. 7C,D). Taken together, we assume that SKD1(E235Q) inhibits the reformation of lysosomes from the hybrid organelle that leads to their accumulation.
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Discussion |
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|
SKD1 regulates the membrane transport via endosomes
The accumulation of LDLR in the E235Q compartments is compatible with the
observations of a previous study showing that LDL uptake is inhibited in
SKD1(E235Q)-expressing CHO cells
(Yoshimori et al., 2000). A
likely explanation for the deficiency in LDL uptake is the absence of LDLR on
the cell surface caused by its sequestration in E235Q compartments. As
endocytosis itself from the PM is not impaired in SKD1(E235Q)-expressing cells
(as measured by dextran, horseradish peroxidase and EGF uptake), it is likely
that the accumulation of the receptors in E235Q compartments is due to the
inhibition of vesicle budding and/or fission from the recycling/sorting
endosomes to the PM (Fig.
8A).
We demonstrated that despite the fact that the TGN integrity was normal, a significant fraction of TGN38 was localized to the E235Q compartments. The accumulation of TGN38 in E235Q compartments is, therefore, due to the similar reason with LDLR and ASGPR, e.g. the inhibition of membrane efflux from recycling/sorting endosomes to the TGN (Fig. 8A).
We further found that expression of SKD1(E235Q) results in the accumulation
of endolyn in the E235Q compartments accompanying with decrease from the PM.
Recent study on endolyn traffic in MDCK cells revealed that a significant
fraction (up to 50%) of newly synthesized endolyn appeared on the cell surface
and about 30% of endolyn endocytosed from the PM are recycling between early
endosomes and the PM (Ihrke et al.,
2001). Thus we assume that a small but considerable fraction of
endolyn is recycling between endosomes and the PM in NRK cells too.
SKD1(E235Q) reduced the cell surface appearance of endolyn (56% of control)
and the internalization of antibody to endolyn from the PM (70% of control).
These reductions are ascribed to the defect in the endolyn recycling from
early endosomes by the expression of SKD1(E235Q). There were still significant
amounts of cell surface appearance and the internalization of endolyn in the
cells expressing SKD1(E235Q). They may be attributed to the supply of newly
synthesized endolyn from the TGN and the subsequent internalization of them
from the PM, which are not abrogated by SKD1(E235Q). Together, our results
suggest that SKD1(E235Q) impairs the membrane traffic out of early and/or
recycling endosomes, leading to the accumulation of molecules that pass
through there (Fig. 8A).
CI-MPR is known to be recycled between the TGN and PM via early and late
endosomes (Mellman, 1996). A
previous study has indicated that expression of SKD1(E235Q) altered the
distribution of CI-MPR from the TGN to the E235Q compartments
(Bishop and Woodman, 2000
).
Since CI-MPR poorly colocalizes with lgp120, a marker for late endosomes and
lysosomes, in the cells expressing SKD1(E235Q), it is believed that CI-MPR is
redistributed to the early rather than to late endosomal E235Q compartments.
Such a redistribution of CI-MPR from the TGN to the E235Q compartments may
lead to the hyper-secretion of lysosomal enzymes (see below). Taken together,
the expression of SKD1(E235Q) impairs membrane transport out of early
endosomes, leading to an accumulation of molecules that are delivered to the
cell surface, the TGN, or late endosomes/lysosomes into early endosomal E235Q
compartments (Fig. 8A). We
conclude that SKD1 is a master molecule regulating the membrane dynamics of
endosomes by its own ATPase activity.
SKD1(E235Q)-expressing cells have a mammalian class E phenotype
SKD1(E235Q) expression blocked the degradation of EGFR and resulted in the
accumulation of both EGF and EGFR in the E235Q compartments. This is analogous
to the inhibition of Ste2p degradation in several yeast class E mutants.
SKD1(E235Q) expression also resulted in a delay in the proteolytic processing
of cathepsin D and induced hyper-secretion of the immature form of this
molecule. Similarly, several class E mutants inhibited the recycling of
Vps10p, which is a receptor for CPY, from pre-vacuolar compartments to the
TGN, which resulted in a delay in the processing and partial secretion of CPY
(Babst et al., 1997). By
analogy, the accumulation of CI-MPR in the E235Q compartments might account
for the mistargeting of cathepsin D. Taken together, we conclude that
SKD1(E235Q) expression in mammalian cells indicates the phenotype closely
related to the class E vps in Saccharomyces cerevisiae.
Class E vps mutants exhibit abnormally enlarged and multilamellar
structures (known as `class E compartments') that accumulate newly synthesized
vacuolar hydrolysase precursors as well as endocytosed plasma membrane
proteins and Golgi-resident proteins (which normally recycle to the Golgi from
the endosomes) (Piper et al.,
1995
; Raymond et al.,
1992
; Rieder et al.,
1996
; Zahn et al.,
2001
). E235Q compartments fully satisfy these criteria.
SKD1(E235Q) impairs the reformation of lysosomes from hybrid
organelles
One of the most pronounced features of SKD1(E235Q)-expressing cells is the
appearance of the enlarged lysosomal compartments. We assume that they are the
accumulated hybrid organelles. Previous studies have suggested the existence
of hybrid organelles in normal cells
(Luzio et al., 2000), which
appear to be derived from a direct fusion of late endosomes with lysosomes
(Bright et al., 1997
;
Mullock et al., 1998
). Their
mean diameter (0.96 µm) is larger than that of either rat liver lysosomes
(0.38 µm) or late endosomes (0.34 µm), and they have a density that is
intermediate between that of late endosomes and lysosomes
(Mullock et al., 1998
). In NRK
cells,
15% of lysosomes appear to be fused with late endosomes to form
hybrid organelles and it was found that lysosomes can be reformed from this
compartment (Bright et al.,
1997
). The following evidence also supports the hypothesis that
the aberrant lysosomes in SKD1(E235Q)-expressing cells are hybrid organelles.
First, the enlarged lysosomes in SKD1(E235Q)-expressing cells contained both
LBPA and cathepsin L. LBPA was originally defined as a specific marker for
late endosomes/MVBs in which it is concentrated within internal vesicles
(Kobayashi et al., 1998
).
Recent studies have revealed, however, that LBPA also localizes in lysosomes
as well as late endosomes/MVBs (Reaves et
al., 2000
). Therefore, although it is not clear whether LBPA can
be used to distinguish late endosomes/MVBs from lysosomes, the fact that LBPA
colocalized almost completely with cathepsin L and pre-loaded TR-dex in
SKD1(E235Q) expressing cells (data not shown) reveal that the aberrant
lysosomes induced by expression of SKD1(E235Q) have characteristics of the
hybrid organelle. Second, the diameter of the enlarged lysosomes (preloaded
with TR-dextran) in SKD1(E235Q)-expressing cells was consistent with that
reported previously (Bright et al.,
1997
). Third, ultrastructural analysis revealed the clustering and
enlargement of MVBs, which occasionally docked and fused with lysosomes. Since
the number of typical lysosomes decreased in the SKD1(E235Q)-expressing cells,
we speculate that despite the fact that fusion of MVBs and pre-existing
lysosomes are not abrogated, the reformation of lysosomes from the hybrid
organelle is inhibited by the expression of SKD1(E235Q). Interestingly, Pryor
et al. showed that the reformation of lysosomes from the hybrid organelle
requires ATP (Pryor et al.,
2000
). Although the authors concluded that proton pump
(ATPase)-dependent acidification is the crucial event requiring ATP, this does
not exclude the possible involvement of SKD1 ATPase activity in the
reformation process.
We cannot rule out the possibility that the enlargement of lysosomes may be
attributed to the secondary effect through the deficiency of either vesicle
transport from or the maturation of, early endosomes. However, two previous
works demonstrated that dysfunction of early endosomes did not alter the
morphology of preexisting lysosomes. While the inactivation of early endosomal
compartments by the crosslinking of endosomal proteins with the biotin-avidin
complex formation caused the partial dysfunction of early endosomes, no
morphological alteration of late endosomes/lysosomes was observed
(Parton et al., 1992). The
enlargement of early endosomes and retardation of membrane transport from
early to late endosomes by overexpression of the invariant chain did not
induce the enlargement of pre-existing lysosomes
(Romagnoli et al., 1993
).
These results imply that early endosome dysfunction itself is not involved in
the enlargement of lysosomes. Our models showing that SKD1 is involved in
multiple membrane transport steps in the mammalian endocytic pathway is in
agreement with a recent study on the yeast homologue of SKD1 (End13p/Vps4p),
which is required for both early-to-late endosome and late endosome-to-vacuole
(Zahn et al., 2001
).
End13/Vps4 mutant cells indicated the accumulation of FM4-64 and
Ste2p in early endosomes as well as the transient accumulation of the
precursor form of CPY in the late endosomes. By analogy to this, it is likely
that SKD1 also functions in multiple membrane transport steps in the endocytic
pathway, not only at a membrane efflux from early endosomes but also at a
lysosome reformation from hybrid organelle
(Fig. 8). Whether SKD1
regulates the late endocytic membrane transport directly or indirectly should
be further tested experimentally in future studies.
The enlarged lysosomal organelle in SKD1(E235Q)-expressing cells resembles
giant lysosomes found in cells from patients with Chediak-Higashi
syndrome (CHS) (White,
1966). We tested whether SKD1 could be involved in the formation
of giant lysosomes in CHS patients by overexpressing the wild-type SKD1
molecule in CHS cells. However, no changes in the frequency or size of the
giant lysosomes in these cells were observed (data not shown). This suggests
that the SKD1 molecule locates upstream of the CHS protein, which is a
lysosomal trafficking regulator denoted as LYST
(Barbosa et al., 1996
) that
participates in the membrane traffic cascade to the lysosomes.
Molecular mechanism of SKD1 function
It is known that SKD1 ATPase activity drives the membrane recruitment of
class E Vps proteins (Babst et al.,
2000; Babst et al.,
1998
; Bishop and Woodman,
2001
). The dominant negative effect of SKD1(E235Q) is most likely
due to the accumulation of the ATP-binding form of SKD1 in the membrane.
Moreover, recently it was shown that a charged MVB protein (CHMP1) was
recruited to E235Q compartments (Howard et
al., 2001
). Thus, we speculate that the overexpression of
SKD1(E235Q), which is a nucleotide bound form of SKD1, inhibits the
dissociation of both class E Vps and CHMP proteins from the membrane and
blocks the subsequent re-utilization of these molecules in the next round of
vesicle formation and/or fission. This hypothesis may account for the absence
of SKD1(E235Q) itself in some of the E235Q compartments (Figs
1,
6). The mutation in another
mammalian class E Vps gene, mouse Vps23/TSG101 indicated the inhibition of
EGFR degradation and induction of hypersecretion of pro-cathepsin D
(Babst et al., 2000
), which are
similar but not identical phenotypes to those of SKD1(E235Q). As
tsg101 cells did not show a change in the morphology of the early
endosomes and the recycling of TfR, the differences in phenotype between
tsg101 and SKD1(E235Q) may suggest that SKD1 regulates the membrane
association of not only class E vps proteins, including Vps23p/TSG101, but
also other cytosolic factors that are responsible for the recycling steps. How
do class E vps proteins regulate endosomal membrane transport? It was recently
shown that the ubiquitin-dependent sorting of carboxypeptidase S into the
inner vesicles of the MVB requires a 350 kDa complex, referred to as endosomal
sorting complex required for transport 1 (ESCRT-1), which comprises Vps23p,
Vps28p and Vps37p (Katzmann et al.,
2001
). They also revealed the ability of Vps23p to bind ubiquitin.
SKD1(E235Q) indeed recruits hVps23p and hVps28p to the E235Q compartments
(Bishop and Woodman, 2001
),
both of which are possible components of mammalian ESCRT-1. Thus,
SKD1-dependent membrane recruitment of ESCRT-1 may regulate the degradation of
EGFR, which is accompanied by ubiqutination-dependent sorting of EGFR from the
limiting membrane to inner membranes of MVBs
(Longva et al., 2002
). Whether
the accumulated EGFR in SKD1(E235Q)-expressing cells is ubiquitinated or not
needs to be determined. Further study of SKD1 and its binding partners is
likely to provide a greater understanding of the mechanisms involved in
membrane transport in the endocytic pathways and the biogenesis of MVBs and
lysosomes.
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Acknowledgments |
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