First published online 4 December 2002
doi: 10.1242/jcs.00213
A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells
Hideaki Fujita1,
Motoko Yamanaka1,
Kanako Imamura1,
Yoshitaka Tanaka1,
Atsuki Nara2,
Tamotsu Yoshimori2,
Sadaki Yokota3 and
Masaru Himeno1,*
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
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Fig. 1. Expression of SKD1(E235Q) induces the accumulation of recycling receptors
in E235Q compartments. MDCK cells were coinfected with adenoviruses encoding
GFP-SKD1(E235Q) and LDLR (A-C). Polarized WIF-B cells were solely infected
with the adenovirus encoding GFP-SKD1(E235Q) and BC indicates the bile
canalicular-like space corresponding to the apical membrane in polarized cells
(D-F). Note that GFP-SKD1(E235Q)-expressing cells (asterisks) had large
phase-lucent vacuoles (C,F). The distribution of GFP-SKD1(E235Q) was
visualized directly by GFP fluorescence (green in A,D). LDLR and ASGPR were
labeled with specific antibodies (B,E) and were merged with GFP images as red
(A,D). Arrows indicate the localization of LDLR (A,B) and ASGPR (D,E) in
basolateral endosomes in the cells that did not express GFP-SKD1(E235Q).
Arrowheads represent the accumulation of LDLR and ASGPR in E235Q compartments
in SKD1(E235Q)-expressing cells. Bars, 20 µm.
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Fig. 6. SKD1(E235Q)-expression induces the accumulation of hybrid organelles.
GFP-SKD1(E235Q)-expressing NRK cells are indicated by asterisks (A-D) and the
localization of GFP-SKD1(E235Q) is directly visualized with GFP (green, A-D).
(A) TR-dextran-labeled lysosomes were enlarged in the cells expressing
GFP-SKD1(E235Q). (B,C) The enlarged lysosomes contained cathepsin L, a
lysosomal enzyme and LBPA, a marker for the late endosomes. (D) Triple
labeling of the cells expressing GFP-SKD1(E235Q) by GFP (green), the
preloading TR-dex (red) and the mAb against lgp120 (blue) was visualized
through a confocal microscope and revealed the heterogeneity of E235Q
compartments (asterisks). Purple arrowheads reveal the co-localization of
lgp120 and TR-dex in the enlarged hybrid organelle. Light blue arrows indicate
the co-localization of lgp120 and GFP-SKD1(E235Q) in the compartments that
lack TR-dex and thus reveal that they are E235Q compartments derived from
either late endosomes or a subset of endosomes that accumulate endocytosed
lgp120. The aberrant structures only labeled with GFP-SKD1(E235Q) represent
E235Q compartments that are separated from late endocytic compartments. (E)
The number and diameter of TR-dex-positive structures were measured in three
separate experiments (in total nine infected cells and 16 uninfected cells).
The size distribution (vesicle diameter, µm) of the TR-dex-positive
structures is represented as a histogram of the mean vesicle number/cell. The
majority of TR-dex-positive structures ranged between 0.2 and 0.8 µm in
diameter in uninfected cells, while the size range of infected cells shifted
between 0.8 and 1.4 µm. Bar, 20 µm.
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Fig. 2. Effects of SKD1(E235Q) expression on TGN38 distribution and traffic. NRK
cells were infected with the adenovirus encoding GFP-SKD1(E235Q), fixed and
stained with mAb against TGN38 (A-C). Panel A shows the merged image of
GFP-SKD1(E235Q) (green) and TGN38 (red). Arrows indicate the colocalization of
GFP-SKD1(E235Q) with TGN38. The cell surface TGN38 was labeled with mAb and
chased at 37°C for 1 hour before fixation and visualization with a
Cy3-labeled secondary antibody (D-F). Panel D shows the merged image of
GFP-SKD1(E235Q) (green) and internalized antibody against TGN38 (red). In
uninfected cells, the internalized antibodies reach the TGN (arrowheads in
D,E), while in infected cells they are colocalized with GFP-SKD1(E235Q) in the
E235Q compartments (arrows in D-F). The integrity of TGN structures was
confirmed with  -adaptin staining (G,H). Asterisks in A, D, G and H show
the GFP-SKD1(E235Q)-expressing cells. Bars, 20 µm.
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Fig. 3. SKD1(E235Q)-expression inhibits the surface appearance of endolyn. NRK
cells expressing GFP-SKD1(E235Q) were fixed and stained with mAb against
endolyn and then with Alexa594-conjugated secondary antibodies (A,B). Panel A
shows the merged image of GFP-SKD1(E235Q) (green) and endolyn (red). Note that
the infected cells (asterisks) exhibited enlarged structures (discussed in
Fig.
6,7).
The cell-surface endolyn was labeled with mAb at 4°C, then fixed
immediately (C,D). Note that the surface expression of endolyn is
significantly reduced in cells expressing SKD1(E235Q) (asterisks in C,D). To
monitor the fate of the internalized mAb, surface-labeled cells were washed
and incubated at 37°C for 1 hour before fixation and then stained with
Alexa594-conjugated secondary antibodies (E,F). Note that the amount of
internalized mAb in infected cells (asterisk in E and F) is less than that in
uninfected cells. They are accumulated into the E235Q compartments in infected
cells while they reach the lysosomes in uninfected cells (E,F). Panel C and E
shows the merged image of GFP-SKD1(E235Q) (green) and the location of
internalized mAb (red). The cell surface binding and the subsequent
internalization of 125I-mAb to endolyn were reduced in the cells
expressing SKD1(E235Q) (G,H), approximately 50 and 70% of controls,
respectively. The amount of 125I-mAb associated to cell surface at
4°C (G) and internalized during the indicated times at 37°C (H) were
represented as the cpm/µg cell protein. Data were obtained from two (G) or
three (H) independent experiments, respectively.
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Fig. 4. EGFR downregulation is impaired by the overexpression of SKD1(E235Q). (A)
GFP-SKD1(E235Q)-expressing and control A431 cells were treated with EGF (500
ng/ml) at 37°C for the periods indicated and the lysates were subjected to
immunoblotting with anti-EGFR or anti-SKD1 antibodies. The 180 kDa band
indicates EGFR, while the 50 kDa and 80 kDa bands indicate endogenous SKD1 and
virally expressed GFP-SKD1(E235Q), respectively. The efficiency of infection
was monitored by the expression of GFP in living cells and usually exceeded
90%. (B) The remaining EGFR bands at each time point were quantitated with
NIH-Image and indicated as a percentage relative to that at time 0 hours. (C)
A431 cells were infected with the GFP-SKD1(E235Q)-expressing adenovirus at a
lower multiplicity of infection, so as to infect about 50% of cells. Then they
were treated with EGF (500 ng/ml) at 37°C for 2 hour and processed for
immunofluorescence analysis with anti-EGFR antibody followed by
Alexa594-labeled secondary antibody. In GFP-SKD1(E235Q)-expressing cells
(asterisks), the accumulation of EGFR in E235Q compartments is shown by yellow
arrowheads. Little or no EGFR is observed in uninfected cells (n.i.).
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Fig. 5. Overexpression of SKD1(E235Q) delays the maturation of cathepsin D and
induces the hypersecretion of pro-cathepsin D. GFP-SKD1(E235Q)-expressing and
normal NRK cells were pulse-labeled with [35S]-methionine/cysteine
for 15 minutes and chased for the periods indicated. The cell lysates and
medium were processed for immunoprecipitation with anti-rat cathepsin D
antibody and the immunoprecipitates were subjected to SDS-PAGE followed by
autoradiography. In the uninfected cells, more than 40% of newly synthesized
pro-cathepsin D (45 kDa) was processed into the mature form (43 kDa) within 1
hour and over 95% within 3 hours. In contrast, in infected cells, more than 90
and 50% of the pro-form remained after 1 and 3 hour of chasing, respectively.
Hypersecretion of pro-form by the infected cells was also observed.
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Fig. 7. Ultrastructural changes in lysosomes and late endosomes in
SKD1(E235Q)-expressing cells. 3Y1-B cells were infected with an adenovirus
encoding GFP-SKD1(E235Q) and processed for immuno-electron microscopic
analysis with the antibody against LGP107 followed by protein-A conjugated to
15 nm gold particles. In control cells (A,B), gold-labeled lysosomes and MVBs
(late endosomes) are indicated by arrows and arrowheads, respectively.
Adenovirus-infected cells (C-F) showed an accumulation and enlargement of
hybrid organelles. The clusters of MVBs are indicated by arrows (C,E,F) and
the enlarged hybrid organelle containing indigestive materials are marked by
asterisks (C,D), respectively. The typical hybrid organelle-like structures
(MVBs directly fused with electron dense lysosomes) are indicated by
arrowheads (A,C,E). Bars, 1 µm.
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Fig. 8. SKD1 functions in mammalian endosomal/lysosomal systems at multiple steps
in membrane transport. A schematic diagram represents the stages of membrane
transport that are inhibited by the overexpression of SKD1(E235Q). (A)
SKD1(E235Q)-expression inhibits the early endosomal recycling of TfR, LDLR and
ASGPR to the PM as well as of TGN38 and MPR to the TGN. EGFR downregulation
was suppressed by the inhibition of either the vesicle transport from early to
late endosomes or the maturation (multivesicular formation?) of the early
endosome into a late endosome. (B) SKD1(E235Q)-expression may inhibit the
reformation of lysosomes from the hybrid organelles. This defect leads to the
accumulation of hybrid organelles.
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© The Company of Biologists Ltd 2003
