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: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 9, 2010
| vol. 107
| no. 45www.pnas.org/cgi/doi/10.1073/pnas.1010554107
con and competes with UVRAG–Rubicon binding, which in turn
activates C-VPS/HOPS activity on Rab7. Finally, depletion of
of EGFR. Hence, Rubicon negatively regulates Rab7 activation
and endosome maturation as a Rab7 effector (Fig. S8).
We have established that GTP-bound Rab7 releases UVRAG
from Rubicon sequestration. There are two consequences of this
UVRAG release (Fig. S6). First, the released UVRAG binds
and activates C-VPS/HOPS (15). Additionally, PI3KC3 activity
may be affected, given that UVRAG is shown to positively reg-
ulate PI3KC3 (21). If that were the case, we would expect that
active Rab7 could stimulate PI3KC3 activity. Interestingly,
overexpressed Rab7 was able to stimulate PI3KC3 and partially
compensate the PI3KC3 deficiency (27), supporting this idea. It
is suggested that p150 interacts with Rab7 to fulfill this function.
However, p150 preferentially binds to an inactive nucleotide free
form rather than active GTP-bound form of Rab7 (27), which we
also confirmed (Fig. 2C). This observation makes it difficult to
explain the stimulation effect of Rab7 on PI3KC3 activation.
Our study provides an alternative model in which GTP-bound
Rab7 abolishes the Rubicon’s sequestration of UVRAG and
allows activation of PI3KC3 by UVRAG (Fig. S8).
UVRAG and Rubicon play critical functions not only in
endosome maturation but also in autophagosome maturation
(18, 19). Emerging evidence suggests that endocytic proteins are
also involved in autophagy regulation (31, 32). Upon autopha-
gosome formation, they fuse with endosomes to form amphi-
somes that eventually fuse with lysosomes or directly fuse with
lysosomes to form autolysosomes. In hepatocytes, there are more
than five times as many amphisomes formed as autolysosomes
(33), suggesting that fusion with the endocytic vesicles could
constitute a major part of autophagosome maturation, at least
in certain cell types. Endosomes have been implicated in auto-
phagosome maturation as a result of the involvement of
ESCRTIII (34, 35) and COPI (36) in autophagy regulation. In
this study, we have provided a molecular linkage connecting
PI3KC3, autophagy, and endocytosis, in which Rubicon serves as
a key negative regulator.
In summary, Rubicon serves as a Rab7 effector that negatively
regulates endosome maturation. Dissection of this molecular
switch will provide important insight into the mechanisms un-
derlying the endocytic and autophagic pathways. Such in-
formation should be critical for the development of novel
therapeutic tools for multiple human pathological conditions that
result from the dysfunction of autophagic and endocytic degra-
Materials and Methods
Cell Culture, Plasmids, and Antibodies. Tetracycline-inducible cell lines were
established, and immunoblotting and immunoprecipitation were performed
as described (16, 37). The full-length cDNA for human Rubicon (KIAA0226)
was generated by PCRs on the basis of a partial cDNA clone purchased from
Kazusa. Additional information is provided in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Jae Jung (University of Southern Califor-
nia, Los Angeles, CA) for UVRAG constructs, Randy Schekman for discussion,
Suzanne Pfeffer for suggestions, and Livia Wilz, Mary Grace Lin, and Jing
Zhang for the critical reading of the manuscript. This work was supported by
a New Investigator Award for Aging from the Ellison Medical Foundation,
the Hellman Family Fund, and National Institutes of Health Grant R01
CA133228 (to Q.Z.).
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