EUKARYOTIC CELL, Apr. 2010, p. 532–538
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 9, No. 4
Intervention of Bro1 in pH-Responsive Rim20 Localization in
Jacob H. Boysen,1† Shoba Subramanian,2† and Aaron P. Mitchell1,2*
Department of Microbiology and Institute of Cancer Research, Columbia University, New York, New York 10032,1and
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 152132
Received 2 February 2010/Accepted 9 February 2010
Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20,
which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with
endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through
proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount
of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP)
under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or
alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT
components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the
localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that
Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the
lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the
expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is
not sufficient to promote Rim101 activation.
The Bro1 domain family consists of endosome-associated
proteins involved in membrane trafficking and signal transduc-
tion (4, 12, 15). The Bro1 domain directs endosomal associa-
tion through interaction with Snf7 (10), a subunit of ESCRT-
III (endosomal sorting complex required for transport). All
eukaryotic genomes encode at least two Bro1 domain protein
family members. Prior studies with Saccharomyces cerevisiae
indicate that its two Bro1 domain proteins, membrane traffick-
ing component Bro1 and alkaline pH signaling protein Rim20,
require Snf7 interaction and endosomal localization for func-
tion (4, 13, 25). In both cases, mutations that abolish interac-
tion with Snf7 result in nonfunctional protein (10, 25).
Bro1 acts as a modular scaffold: the N-terminal Bro1 domain
interacts with Snf7 and tethers Bro1 to the endosome, where
its C-terminal region recruits the ubiquitin protease Doa4 in
proximity to ubiquitinated multivesicular body cargo (1, 10, 14,
19). Molecular and sequence analyses indicate that Rim20 also
functions as a modular scaffold (4, 24, 25). Rim20, like its
Aspergillus nidulans ortholog PalA, facilitates the proteolytic
activation of the transcription factor Rim101 (whose A. nidu-
lans ortholog is PacC), a pH-responsive zinc finger transcrip-
tion factor that is highly conserved across fungal species (4, 5,
16, 24, 25). Rim101 proteolysis is dependent upon the cysteine
protease Rim13, putative membrane proteins Rim9, Rim21,
and Dfg16, and a soluble arrestin-like molecule, Rim8 (2, 7,
20). Rim101 proteolytic processing also requires Snf7, as well
as subunits of the ESCRT-I (Vps23, Vps28, Vps37), ESCRT-II
(Vps36, Snf8, Vps25), and ESCRT-IIIA complex (Vps20, Snf7).
The ESCRT subunits required for Rim101 processing are also
required for Snf7 recruitment to endosomes, while ESCRT
subunits that do not affect Snf7 localization are not required
for Rim101 processing (4, 25).
Despite their broad similarity, functional analysis has not
found a significant link between Rim20 and Bro1 (3, 15, 24).
While the localization of both Bro1 and Rim20 depends on
ESCRT, distinct upstream inputs also exist. Bro1 robustly lo-
calizes to endosomes under acidic conditions, while very little
Rim20 localizes to endosomes under similar conditions (4).
Proper Rim20 localization, but not Bro1 localization, requires
the Rim101 putative signaling complex (Rim8, Rim9, Rim21,
and Dfg16), as well as an alkaline environment, suggesting the
existence of distinct endosome compartments for each Bro1
domain protein. Colocalization analysis supports this view:
some endosomes are associated with only Bro1 or Rim20 (4).
Interestingly, environmental pH levels may influence not only
Rim20 but also Bro1 localization, as absolute levels of Bro1
foci fall under alkaline conditions. Thus, as levels of one Bro1
domain protein associated with an ESCRT-endosome popula-
tion rises, the levels of the homologous protein associated
with an ESCRT-endosome population falls (4). Here we have
tested the specific model that Bro1 and Rim20 compete for
association with ESCRT-endosomes. Our findings indicate
that Bro1-Rim20 competition occurs in vivo, but functional
analysis argues that competition is not a major pH response-
MATERIALS AND METHODS
Strains and plasmids. The S. cerevisiae strains used here are listed in Table 1,
and the primers used are listed in Table 2. The bro1?::URA3 mutation was
created by PCR-directed gene disruption using primers BRO1.URA3 F and
BRO1.URA3 R against a pRS306 template and transformed and URA?cells
were selected. Disruptions were confirmed by PCR using primers flanking the
target region. Overexpression plasmids encoding BRO11-367(pJB31) and BRO1
* Corresponding author. Mailing address: Department of Biological
Sciences, Carnegie Mellon University, 4400 Fifth Avenue, MI 200B,
Pittsburgh, PA 15213. Phone: (412) 268-5844. Fax: (412) 268-7129.
† Contributed equally.
?Published ahead of print on 26 February 2010.
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538BOYSEN ET AL.EUKARYOT. CELL