Rubicon controls endosome maturation as
a Rab7 effector
Qiming Sun, Wiebke Westphal, Kwun Ngok Wong, Irena Tan, and Qing Zhong1
Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved October 6, 2010 (received for review July 21, 2010)
The activation and recruitment of the small GTPase Rab7 to early
endosome is a critical step for early to late endosome maturation,
a process that requires the class III phosphatidylinositol 3-kinase
(PI3KC3) and GTPase regulators. However, the molecular mecha-
nism underlying Rab7 activation and endosome maturation is still
poorly defined. Here we report that Rubicon, a component of the
PI3KC3 complex, prevents endosome maturation through differen-
tial interactions with Rab7 and UVRAG. UVRAG activates PI3KC3
and C-VPS/HOPS, a guanine nucleotide exchange factor that cata-
lyzes the exchange of GDP for GTP on Rab7. We demonstrate that
Rubicon sequesters UVRAG from C-VPS/HOPS. Active GTP-bound
Rab7 competes for Rubicon binding and releases UVRAG to associ-
ate with C-VPS/HOPS, which in turn promotes further loading of
Rab7 with GTP. This feed-forward loop ensures rapid amplification
of GTP-bound Rab7 and consequent stimulation of endosome mat-
uration. Hence, Rubicon serves as a previously unknown Rab7 ef-
fector to ensure the proper progression of the endocytic pathway.
autophagy|endocytosis|epidermal growth factor|autophagosome|
specific Rab GTPase proteins (1). In the endocytic pathway, early
endosomes are characterized by the small GTPase Rab5, early
endosomal antigen EEA1, and PI3KC3 (2–4). Rab5 regulates
homotypic fusion of early endosomes. These early endosomes
then mature into late endosomes, which will be targeted for ly-
sosomal degradation or processing (5). Understanding the mo-
lecular mechanisms underlying endosome maturation is of
clinical significance, as many bacteria (e.g., Mycobacterium tuber-
culosis) and viruses (e.g., HIV) interfere with phagolysosomal or
endolysosomal maturation to avoid elimination in lysosomes (6).
The early-to-late endosome transition begins with the activa-
tion and recruitment of Rab7 to the subdomains of early endo-
somes bearing Rab5, followed by Rab5 displacement from the
same vesicle (5, 7) or endosome fission with the formation of late-
endosome-targeted transportation vesicles (8). The activation of
Rab7 is required for endosome maturation. However, little is
known about how Rab7 activation is controlled.
As a small GTPase, Rab7 regulates membrane trafficking by
cycling between inactive (i.e., GDP-bound) and active (i.e., GTP-
and vacuole protein sorting) complex is the guanine nucleotide
GTP transition, leading to Rab7 activation. Active GTP-bound
Rab7 binds to Rab7 effectors and executes its function in vesicle
tethering, docking, and fusion (10, 11). The GTPase activating
proteins (GAP) inactivate Rab7, stimulating the conversion
from the GTP-bound form to the GDP-bound form (12, 13). The
of active GTP-bound Rab7 (14). Recent evidence indicates that
this complex is positively regulated by UVRAG, a component of
PI3KC3 (15), which provides an interesting link between PI3KC3
and Rabs that are both crucial for endosome maturation.
We and others have recently purified a PI3KC3 holocomplex
that includes hVPS34, p150, Beclin 1, UVRAG, and Barkor/
ndocytic transport involves the passage and sorting of cargo
within different sets of vesicles that are marked by organelle-
Atg14(L) (16–20). PI3KC3 forms two mutually exclusive pro-
tein subcomplexes that localize to autophagosome or endosome
and execute distinct functions. The autophagosomal complex is
composed of the PI3KC3 core complex (hVPS34, p150, and
Beclin 1) and Barkor/Atg14(L). Barkor/Atg14(L) is the targeting
factor for this subcomplex to nascent autophagosomes (16–20).
The endosomal complex consists of the PI3KC3 core complex
and UVRAG. UVRAG positively regulates PI3KC3 activity and
is required for autophagosome and endosome maturation prob-
ably via its direct interaction with C-VPS/HOPS (15, 21). The
endosomal complex also contains Rubicon (Run domain protein
as Beclin 1 interacting and cysteine-rich containing), which serves
as a negative regulator of autophagosome maturation (18, 19).
Although it has been suggested that Rubicon plays a negative role
in endosome maturation, this function is still under debate.
Here we report that Rubicon is a key negative regulator in
endosome maturation and that active Rab7 antagonizes this
inhxibition. Rubicon is highly enriched on Rab5-positive early
endosomes and sequesters UVRAG from C-VPS/HOPS. Active
GTP-bound Rab7 competes for Rubicon binding and releases
UVRAG. This release promotes the complex formation between
UVRAG and C-VPS/HOPS to further activate Rab7. These
eventstrigger andamplifytheearly-to-late endosomematuration.
Rubicon Interacts with Rab7 via Its C Terminus. We recently iden-
tified a Beclin 1/PI3KC3 complex using Beclin 1 as bait in human
osteosarcoma U2OS cells (16). This complex was composed of
the PI3K catalytic subunit hVps34, p150 regulatory subunit,
UVRAG, and Barkor/Atg14(L). In the same complex, Rubicon
(also called p120 or Baron) was also identified (16, 18–20).
Rubicon contains 972 amino acids, with a recognizable RUN
domain (named after RPIP8/UNC-14/NESCA) that is shared by
a group of proteins interacting with small GTPases (22–24).
Given Rubicon’s RUN domain and the importance of Rab5 and
Rab7 in endosome maturation, we tested whether Rubicon inter-
acts with the Rab family. Rab7, but not Rab5, coimmunoprecipi-
tated with Rubicon (Fig. 1A). As Rubicon is known to associate
with UVRAG in the PI3KC3 complex (18, 19), we investigated
whether Rab7 coexists with UVRAG in the same protein complex.
Beclin 1, Vps16 (a component of C-VPS/HOPS complex), Rab7,
and Rubicon. UVRAG consistently interacted with the PI3KC3
components including Vps34, Beclin 1, and Rubicon (Fig. 1B).
UVRAG also associated with Vps16 (Fig. 1B). However, no Rab7-
UVRAG interaction could be detected in this coimmunoprecipi-
tation assay. Therefore, Rab7 and UVRAG form distinct com-
plexes with Rubicon.
Author contributions: Q.S. and Q.Z. designed research; Q.S., W.W., K.N.W., and I.T.
performed research; Q.S. and Q.Z. analyzed data; and Q.S. and Q.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 9, 2010
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We further refined the interaction domain between Rubicon
and Rab7. A series of Rubicon deletion mutants were generated,
including mutants lacking the RUN, FYVE-like, or coiled-coil
domains (CCD) from full-length Rubicon. We also created three
large polypeptide fragments containing the RUN, CCD, or
FYVE-like domains (Fig. 1C). The interaction of these mutants
with Rab7 was evaluated by Rab7 immunoprecipitation assay.
Full-length Rubicon, rather than vector alone, interacted with
Rab7 (Fig. 1C, lanes 1–2). The RUN and CCD domains were
dispensable for Rab7 interaction (Fig. 1C, lanes 3–4 and 6–7),
whereas a C-terminal (CT) region (aa 880–972) was required
(Fig. 1C, lane 5). A larger C terminus region of Rubicon (aa 600–
972) containing the CT domain was sufficient for Rubicon to
interact with Rab7 (Fig. 1C, lane 8).
Rubicon Preferentially Binds to GTP-Bound Rab7. Rab7 regulates
membrane trafficking by cycling between inactive (i.e., GDP-
bound) and active (i.e., GTP-bound) conformations (25, 26). We
then tested whether the guanine nucleotide cycling of Rab7 is
crucial for its interaction with Rubicon. Coimmunoprecipitation
experiments in 293T cells showed Rubicon binding to WT Rab7.
Rab7 exhibited a relatively stronger binding to a constitutively
active GTP-bound Rab7 Q67L mutant protein and much weaker
binding to a GDP-bound Rab7 T22N mutant protein (Fig. 2A).
To further confirm that the GTP-loaded Rab7 binds to Rubicon,
we performed a coimmunoprecipitation experiment in the pres-
ence of excess amount of nonhydrolyzable GTP (GTP-γ-S) or
GDP (GDP-β-S). The loading of GDP (GDP-β-S), rather than
GTP (GTP-γ-S), decreased the Rab7–Rubicon interaction (Fig.
S1). These results suggest that Rubicon could preferentially bind
to Rab7 in GTP-bound form rather than GDP-bound form.
To test for a direct interaction between Rab7 and Rubicon in
a nucleotide-dependent manner, we expressed and purified re-
combinant HA-tagged Rab7 WT, Q67L, and T22N mutants from
Escherichia coli (Fig. S2A). We also expressed and purified
Myc (lanes 1–3) or Rab7-Myc vectors (lanes 4–6). The whole 293T cell lysates (lanes 1 and 4) were immunoprecipitated with anti-Flag antibody (lanes 3 and 6)
or control IgG (lanes 2 and 5), followed by immunoblotting with anti-Flag antibody for Rubicon and anti-Myc antibody for Rabs. (B) Rubicon interacts with
UVRAG and Rab7 in different complexes. Myc tagged Vps34, Beclin 1, Vps16, Rab7, and Rubicon were expressed in HEK293T cells and immunoprecipitated (IP)
with anti-Myc antibody. The proteins indicated were detected by immunoblotting (IB) in the bound (B) and input fractions. (C) The CT domain of Rubicon is
required for Rab7 binding. A vector expressing HA-tagged Rab7 was cotransfected with EGFP-Rubicon-Flag (WT) vector or the vectors expressing different
mutants, followed by immunoprecipitation with anti-HA antibody (Rab7) and immunoblotting with the antibodies indicated.
Rubicon interacts with Rab7 via its C terminus. (A) Rubicon interacts with Rab7 but not Rab5. Rubicon-Flag vector was cotransfected with either Rab5-
cells with Rab7-Myc vectors expressing Rab7-WT, GTP-bound Rab7 Q67L mutant, or GDP-bound Rab7 T22N mutant, followed by immunoprecipitation with
anti-Myc antibody (Rab7) and immunoblotting with the antibodies indicated. (B) Rab7 differentially interacts with Rubicon and p150. Purified recombinant
HA-tagged Rab7 (0.1 μM) was incubated with recombinant Flag-tagged p150, Vps34, Beclin 1, UVRAG, Barkor/Atg14(L), or Rubicon (0.1 μM) in an in vitro pull-
down assay using HA affinity beads. The bound and input proteins were detected by anti-Flag or HA antibodies. (C) Rubicon directly binds GTP-bound Rab7.
Recombinant full-length Flag-tagged p150, Vps34, or Rubicon (0.1 μM) were incubated with HA affinity beads prebound with recombinant HA-tagged Rab7
WT, T22N, or Q67L mutant proteins (0.1 μM). The bound proteins were resolved by 7.5% SDS/PAGE and analyzed by Western blot using anti-Flag antibody.
Rubicon preferentially binds to GTP-bound Rab7. (A) Rubicon binds to GTP-bound Rab7 in vivo. Rubicon-Flag vector was cotransfected in HEK293T
Sun et al. PNAS
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recombinant full-length Flag-tagged Vps34, UVRAG, Beclin 1,
Barkor/Atg14(L), and Rubicon from baculovirus-infected insect
Rab7. We then performed a pull-down assay with all purified
components and observed that both p150 and Rubicon, but not
other PI3KC3 components, bind directly to Rab7 (Fig. 2B). We
further tested the binding of different forms of Rab7 proteins to
p150 and Rubicon. The binding of p150 to WT Rab7 was clearly
detected (Fig. 2C). p150 bound to GDP-bound Rab7 T22N much
more weakly; essentially no binding of p150 to the GTP-bound
Rab7 Q67L mutant was observed (Fig. 2C). This result is consis-
tent with the published observation that p150 preferentially
interacts with nucleotide-free Rab7 (27). In contrast to p150,
Rubicon preferentially interacted with GTP-bound Rab7 (Fig.
2C), suggesting that it might function as a Rab7 effector via
Rab7 Competes with UVRAG for Rubicon Binding. Rubicon interacts
with UVRAG and Rab7 in distinct protein complexes (Fig. 1B).
We speculated that Rab7, especially the GTP-bound form, might
then compete with UVRAG for Rubicon binding. Thus, we
overexpressed UVRAG to test its effect on Rubicon–Rab7 in-
teraction. UVRAG overexpression dramatically decreased the
amount of Rubicon coimmunoprecipitated with Rab7 or Rab7
pulled down by Rubicon (Fig. 3A). Conversely, Rab7 over-
expression also compromised the UVRAG–Rubicon interaction
in the immunoprecipitation assay (Fig. 3B). These results suggest
that Rab7 and UVRAG compete for interaction with Rubicon in
vivo. To test if Rab7 competes with UVRAG for Rubicon binding
in a nucleotide-dependent manner, Rab7 WT, GDP-bound T22N
mutant, or GTP-bound Q67L mutant proteins were expressed
andtestedfortheir effects on Rubicon–UVRAG interaction. The
GTP-bound Rab7 Q67L mutant competed with UVRAG for
Rubicon binding much more efficiently than the GDP-bound
Rab7 T22N mutant (Fig. 3C). We therefore concluded that GTP-
bound Rab7 competes with UVRAG for Rubicon binding in vivo.
We further tested the direct competition between Rab7 and
UVRAG for Rubicon binding in vitro. We expressed and purified
recombinant full-length GST-tagged Rab7 WT, GDP-bound
(Fig. 3D) and incubated these proteins with the preassembled
Rubicon–UVRAG complex in a purified system. Rab7 Q67L
highest efficiency, whereas the GDP-bound Rab7 T22N had al-
Rubicon from UVRAG in a nucleotide-dependent manner.
Rubicon Is Highly Enriched on Rab5-Decorated Early Endosomes. To
explore the potential role of Rubicon in endosome organization,
U2OS cells stably expressing Flag-Rubicon. We generated a cell
line that inducibly expresses Flag-Rubicon under the control of
doxycycline. When Flag-Rubicon expression was induced by an
extremely low dose of doxycycline (1 ng/mL), Flag-Rubicon was
Flag-Rubicon expressed at physiological level displayed a signifi-
cant overlap with early endosome markers EEA1, UVRAG, and
FYVE2 (Fig. S3B). We then tested Rubicon colocalization with
Rab7 in Rubicon-expressing cells. Only limited colocalization
between Rubicon and Rab7 could be observed in cells expressing
Rubicon at a physiological level. No colocalization was detected
between Rubicon and an inactive Rab7 T22N GDP-bound form
(Fig. S3C). Rab7 colocalization with Rubicon was only apparent
when a constitutively active GTP-bound Rab7 Q67L form was
Rubicon preferentially binds to active GTP-bound Rab7.
We next compared the localization of Rubicon expressed at
a physiological level with Rab5 and two Rab5 mutants, GDP-
bound S34N and GTP-bound Q79L. In cells costained for Ru-
bicon and Rab5 (Fig. 4A, Top), Rubicon significantly overlapped
with Rab5 in large cellular puncta. Most Rubicon signal colo-
concentrations of UVRAG were overexpressed in HEK293T cells (Top), and the resulting cell lysates were immunoprecipitated with anti-Rab7 antibody
(Middle) or anti-Rubicon antibody (Bottom). The Rab7 or Rubicon immunocomplex was then probed for the proteins indicated. The normalized binding of
Rubicon and Rab7 was quantified as the relative ratio of bound/input (B/I). The B/I in the control cells was set as 1.0. (B) Overexpression of Rab7 decreases
UVRAG–Rubicon interaction. Rab7 was expressed in HEK293T cells. The resulting cell lysate was then immunoprecipitated with anti-Rubicon antibody. The
bound and input fractions were probed for the proteins indicated. (C) GTP–Rab7 competes Rubicon from UVRAG in vivo. Different forms of Rab7 (WT, T22N,
Q67L) were expressed and the resulting cell lysate was immunoprecipitated with anti-UVRAG antibody. The bound and input were probed for the proteins
indicated. (D) Recombinant purified GST-tagged Rab7 WT and mutants from E. coli were analyzed by 10% SDS/PAGE followed by Coomassie blue staining. (E)
In vitro competition among Rubicon, UVRAG, and different forms of Rab7. Rubicon-Flag-His (0.1 μM) was bound to Ni column first and then incubated with
UVRAG-Flag (0.1 μM) to form a Rubicon–UVRAG complex. Different concentrations (0.1, 0.5, 2 μM) of Rab7 proteins were incubated with the Rubicon–UVRAG
complex. The bound proteins were analyzed by Western blotting using anti-Flag and anti-Rab7 antibodies.
GTP-bound Rab7 competes with UVRAG for Rubicon binding. (A) Overexpression of UVRAG compromises the Rab7–Rubicon interaction. Different
| www.pnas.org/cgi/doi/10.1073/pnas.1010554107 Sun et al.
calized or was detected adjacent to Rab5. However, Rubicon
puncta did not overlap with the GDP-bound S34N form of Rab5
(Fig. 4A, Middle). Expression of the Rab5 GTP-bound Q79L
mutant stimulates homotypic fusion ofearly endosomes and leads
to formation of large ring-like membrane structures (28). These
enlarged endosomes are considered arrested at the convergent
point of endosome maturation after acquiring Rab7, as Rab5
cannot be displaced as a result of inefficient GTP hydrolysis (5, 8,
29). In Rab5 Q79L-expressing cells, Rubicon was highly enriched
in subdomains of large ring-like, Rab5-positive early endosomal
structures (Fig. 4A, Bottom). These data showed that Rubicon
localizes to enlarged early endosomes and subdomains of ma-
To confirm that Rab7 competes with UVRAG for Rubicon
binding in vivo, we expressed GTP-bound Rab7 Q67L or GDP-
bound Rab7 T22N and scored their effect on the colocalization
of Rubicon and UVRAG. In the control mock-transfected cells,
Rubicon colocalized well with UVRAG (Fig. 4B, Top, and 4C).
The colocalization of these two proteins were not altered upon
GDP-bound Rab7 T22N expression (Fig. 4B, Middle, and 4C),
but significantly reduced upon GTP-bound Rab7 Q67L expres-
sion (Fig. 4B, Bottom, and 4C). This further supports our hy-
pothesis that Rab7 GTP-bound form competes for Rubicon
binding with UVRAG.
Rubicon Sequesters UVRAG from C-VPS/HOPS and Blocks Rab7
Activation. UVRAG interacts with C-VPS/HOPS and activates its
GEF activity, resulting in Rab7 activation (15). We speculated that
activation. We tested the UVRAG-Vps16 (a C-VPS/HOPS com-
ponent) interaction in Rubicon RNAi-depleted cells. The in-
teraction between UVRAG and Vps16 was significantly increased
in the absence of Rubicon (Fig. 5A). Conversely, in Rubicon-
(Fig. 5B). This demonstrates that Rubicon sequesters UVRAG
from C-VPS/HOPS interaction.
We hypothesize that Rubicon sequesters UVRAG from C-
VPS/HOPS interaction to block Rab7 activation. If this is true,
Rubicon depletion should lead toRab7 activation,which couldbe
detected by the enhanced interaction between the active GTP-
bound Rab7 and Rab7 effectors. To test this hypothesis, we ex-
amined the interaction between Rab7 and a well known Rab7
effector RILP (Rab-interacting lysosomal protein) (30). RILP
specifically interacts with GTP-bound Rab7; the amount of RILP
coprecipitated with Rab7 could reflect the GTP-binding and ac-
tivation of Rab7. In Rubicon-depleted cells, the association be-
tween Rab7 and RILP was dramatically increased (Fig. 5C),
suggesting that Rab7 is activated. However, it is also possible that
the loss of Rubicon frees up more GTP-bound Rab7 for RILP
binding rather than Rab7 activation. To test this possibility, we
expressed a constitutively active GTP bound Rab7 Q67L mutant
in WT and Rubicon-depleted cells. Rab7 Q67L strongly bound
to RILP but no further increase could be observed in Rubicon-
depleted cells, whereas the Rab7–RILP interaction was clearly
increased in the absence of Rubicon (Fig. S4A), further sup-
porting the notion that the generation of GTP-bound Rab7 is
probably up-regulated without Rubicon.
The interaction between Rab7 and another Rab7 effector,
Vps41 (14), was also investigated. Consistently, the interaction
between Rab7 and Vps41 was dramatically increased in Rubicon-
depleted cells (Fig. 5D). The increased interactions of Rab7–
Vps41 and UVRAG–Vps16 in Rubicon-depleted cells could be
efficiently suppressed by the complementation of RNAi-resistant
Rubicon (Fig. S4B), excluding the off-target effect of RNA in-
terference. Furthermore, overexpression of Rubicon led to a re-
duced Vps41–Rab7 interaction (Fig. S5). Thus, Rubicon sup-
presses Rab7 activation through C-VPS/HOPS, illustrated by
the increased interaction between Rab7 and Rab7 effectors in
Rubicon-deficient cells. This conclusion is further supported by
the translocation of Rab7 from perinuclear regions to cytosolic
puncta in Rubicon-depleted cells (Fig. S6).
As UVRAG is a component of PI3KC3 through its interaction
with Beclin 1 (21), we also investigated if Rubicon sequesters
transfected with GFP-Rab5-WT, GFP-Rab5-S34N, or GFP-Rab5-Q79L and observed under a fluorescence microscope. Cy3-conjugated M2 antibody was used to
label Rubicon-Flag. (Scale bar: 5 μm.) Framed areas of Rab5 Q79L-expressing cells are enlarged (Bottom). (B) Rab7 GTP-bound form disrupts the overlap of
Rubicon–UVRAG. Mock transfection: GDP-bound Rab7 T22N or GTP-bound Rab7 Q67L were coexpressed with GFP–UVRAG and Rubicon-Flag in U2OS cells. The
subcellular localization of UVRAG (green) and Rubicon (red) was observed under a fluorescence microscope. (C) The number of cellular puncta positive for
UVRAG and Rubicon was counted and plotted in cells described in B.
Rubicon localizes to maturing early endosomes. (A) Rubicon localizes to maturing early endosomes. U2OS cells expressing Rubicon-Flag were
Sun et al. PNAS
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UVRAG from Beclin 1. Interestingly, the interaction between
UVRAG and Beclin 1 or Vps34 was largely unaffected in the
presence of Rab7 or Rubicon overexpression (Fig. S7), suggesting
no competition between UVRAG–Beclin 1 interaction and
UVRAG–Rubicon interaction, which is consistent with coex-
To test if Rab7 activation in Rubicon-depleted cells is dependent
on PI3KC3 activity, Rubicon-depleted cells were treated with
aPI3KC3inhibitor,3-methyladenine (3-MA).The 3-MAtreatment
had no effect on Rab7–RILP interaction (Fig. 5C), suggesting that
Rab7 is activated in a PI3KC3-independent manner.
Rubicon Controls Endosome Maturation and Endocytic Degradation.
Rubicon sequesters UVRAG from C-VPS/HOPS binding and
blocks Rab7 activation. Given the essential role of Rab7 in en-
dosome maturation, we therefore anticipated a negative role of
Rubicon in the endocytic degradation pathway. We monitored the
endocytic degradation of epidermal growth factor (EGF) receptor
(EGFR) in the Rubicon-depleted cells. EGF was used to stim-
ulate EGFR activation and subsequent degradation. In Rubicon-
depleted cells, EGFR degradation was accelerated compared with
what is observed in Rubicon WT cells (Fig. 6 A and B). EGFR
turnover was significantly slowed in Rubicon-overexpressing cells
(Fig. 6 C and D). These data further confirm the negative regula-
tion of EGFR degradation by Rubicon.
We complemented Rubicon-knockdown cells with various
RNAi-resistant cDNAs for Rubicon WT or mutant forms and
monitored EGFR degradation. WT Rubicon was able to rescue
the accelerated EGFR degradation, whereas vector alone did not
(Fig. 6 E and F). Importantly, the Rubicon CT deletion mutant
that disrupts the interaction with Rab7 (Fig. 1C) failed to com-
plement Rubicon defects (Fig. 6 E and F). Additionally, in Ru-
bicon overexpression cells, EGFR was accumulated in the large
vacuoles (Fig. 6 G and H). These results demonstrate that the
Rab7 interaction is indispensable for Rubicon’s function in the
in endocytic trafficking, which is to control the endosome matu-
ration by regulating Rab7 and UVRAG. Several lines of evidence
support this conclusion. First, Rubicon interacts with Rab7
and UVRAG in a mutually exclusive manner. Rubicon binds
to UVRAG and sequesters it from the C-VPS/HOPS complex.
Furthermore, GTP-bound Rab7 selectively interacts with Rubi-
lates Rab7 activation. (A) Rubicon depletion promotes the interaction be-
tween UVRAG and C-VPS/HOPS. Cell lysates from Rubicon WT or knockdown
(KD) U2OS cells were immunoprecipitated with anti-UVRAG antibody. The
indicated proteins were detected. (B) Rubicon overexpression decreases the
binding of UVRAG to C-VPS/HOPS. HEK293T cells were transfected with
Rubicon-Myc or vector alone. The cell lysates were immunoprecipitated with
anti-UVRAG antibody. The indicatedproteinswere detected. (C) Depletion of
Rubicon stimulates Rab7-RILP interaction. In cell lysates prepared from WT or
Rubicon RNAi-depleted cells, Rab7 was immunoprecipitated and the result-
ing immunocomplexes were probed for Rab7 and RILP. 3-MA (10 mM) was
usedtotreatboth Rubicon WTanddepleted cellsfor8h beforecollection. (D)
Depletion of Rubicon promotes Rab7–Vps41 interaction. In cell lysates pre-
pared from WT or Rubicon RNAi-depleted cells, Rab7 was immunoprecipi-
tated and the resulting immunocomplex was probed for Rab7 and Vps41.
Rubicon sequesters UVRAG from C-VPS/HOPS and negatively regu-
was examined in Rubicon RNAi-depleted 293T cells. Cells were serum-starved in DMEM-only medium for 10 h, followed by 200 ng/mL EGF addition to initiate
EGFR degradation. Cells were collected at indicated time points and tested for EGFR level by Western Blotting. (B, D, and F) EGFR levels in A, C, and E were
quantified by a Phosphorimager and normalized by calculating as the percentage of the initial receptor content. (C) EGFR degradation was tested in Rubicon
overexpression cells. (E) EGFR degradation was examined in Rubicon-depleted 293T cells expressing vector alone, Rubicon WT, or Rubicon-ΔCT. (G) EGFR
accumulates inlarge vacuolesin Rubicon-overexpressing cells. In U2OS cells expressing vector alone or Rubicon, the endogenousEGFR localization was detected
by immunostaining using anti-EGFR antibody. (Scale bar: 5 μm.) (H) Number of vacuoles greater than 800 nm in diameter was counted and plotted in cells (G).
Depletion of Rubicon alters EGFR degradation via endocytic pathway. (A) EGFR degradation is accelerated in Rubicon-depleted cells. EGFR degradation
| www.pnas.org/cgi/doi/10.1073/pnas.1010554107 Sun et al.