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.
(pJB30) were made by cloning a PCR product encompassing 500 bp of 5?
regulatory sequence and relevant sequence into the pYES2.1 vector (Invitrogen)
in accordance with the manufacturer’s instructions. The Bro1 domain was de-
fined as residues 1 to 367 (bp 1 to 1101), consistent with structural information
Media and growth conditions. Yeast growth media (YPD and SC) were of
standard composition (8). For pH exposure assays with S. cerevisiae, SC medium
containing 0.1 M HEPES was freshly titrated to pH 4.0 with HCl or to pH 8.3
with NaOH and used immediately as described previously (4). All cultures and
plates were incubated at 30°C.
Microscopy. Imaging was performed at room temperature with a Nikon
Eclipse E800 wide-field fluorescence microscope, a Nikon Plan Apochromat
100? 1.4 objective (Nikon, Melville, NY), and a Hamamatsu Orca100 digital
charge-coupled device camera (Hamamatsu, Bridgewater, NJ). Images were
acquired with OpenLab Improvision software and processed in Adobe Photo-
shop CS3 10.01 (Adobe, San Jose, CA) and ImageJ (NIH, Bethesda, MD).
Quantitative PCR (qPCR) analysis. Yeast strain BY4741 and rim101?, bro1?,
dfg16?, bro1? dfg16?, and vps4? mutants were grown overnight, diluted into
fresh YPD to an optical density at 600 nm (OD600) of ?0.2, and grown at 30°C
with shaking to an OD600of ?1.0. Equal amounts of cells were shifted for 120
min to YPD plus 0.1 M HEPES (pH 4.0 or 8.3). Cells were isolated by filtration,
flash-frozen, and stored at ?80°C. RNA isolation and subsequent qPCR analysis
were done as described previously (18). Briefly, total RNA was isolated by using
the RiboPure Yeast kit (Ambion) in accordance with the manufacturer’s instruc-
tions. cDNA was prepared using an AffinityScript Multiple Temperature cDNA
synthesis kit (Stratagene) by following the manufacturer’s instructions. Primers
located in the 3? end of the gene were designed using Primer3 (http://frodo.wi
.mit.edu/primer3/). Reverse transcription (RT) reaction mixtures were prepared
in triplicate using iQ SYBR supermix (Bio-Rad), and RT-PCR was performed
using a Bio-Rad iCycler (Bio-Rad). Data analysis was conducted using Bio-Rad
iQ5 software 2.0. Transcript levels were normalized against TDH3 expression,
and gene expression changes were calculated by the ??CT method.
Competition between Bro1 domain proteins Bro1 and
Rim20 for endosomal localization. Bro1 and Rim20 require
ESCRT-III subunit Snf7 for localization but show a reciprocal
dependence on the environmental pH (4). A simple model is
that one Bro1 domain protein displaces the other from Snf7
and hence allows endosomal localization. Therefore, we rea-
soned that loss of Bro1 may permit Rim20-endosome associ-
ation under otherwise restrictive conditions. We examined the
localization of a functional Rim20-green fluorescent protein
(GFP) fusion, expressed from the RIM20 genomic locus, in a
bro1? mutant background. Under acidic conditions, fewer than
10% of wild-type (WT) control cells exhibited a single Rim20-
GFP focus (Fig. 1A and D). In contrast, all bro1? mutant cells
exhibited one or more Rim20p-GFP foci (Fig. 1C and D). The
positive control vps4? mutant cells (4) also exhibited Rim20-
GFP on endosomes under acidic conditions (Fig. 1B). These
TABLE 1. Strains used in this study
StrainGenotype Reference or source
MATa his3?1 leu2?0 met15?0 ura3?0
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 vps4?::URA3
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 vps27::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 vps23::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 vps25::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 vps20::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 snf7::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 rim8::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 rim9::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 rim13::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 rim20::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 rim21::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 dfg16::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::URA3 rim101::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 rim101::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 dfg16?::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 bro1?::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 vps4?::KANMX4
MATa his3?1 leu2?0 met15?0 ura3?0 RIM20-GFP-HISMX6 dfg16?::KANMX4 bro1?::URA3
ATCC 201388 (BY4741)
TABLE 2. Primers used in this study
RIM8 R JB .........................GATCTCGTCGTTCCTATGAC
RIM8 F JB..........................GCTACGTATGGTCAATCCAG
SMP1 R JB .........................GTTTGTCGAACTCGTGGA
SMP1 F JB..........................GATGTGGCAGCACTTATG
TDH3 R JB.........................TACCAGGAGACCAACTTGAC
TDH3 F JB.........................GAACAAGGAAACCACCTACG
VOL. 9, 2010Bro1 DOMAIN PROTEIN COMPETITION533
findings indicate that Bro1 prevents Rim20-endosome associ-
ation under acidic growth conditions.
A second prediction of the Bro1-Rim20 competition model
is that overexpression of Bro1 will inhibit Rim20-endosome
association under alkaline growth conditions. We quantified
Rim20-GFP foci in cells carrying plasmids that overexpress the
full-length BRO1 gene or a truncated BRO1 sequence encod-
ing only the Bro1 domain. In control cells carrying the plasmid
vector, Rim20-GFP foci were abundant under alkaline condi-
tions (Fig. 2A and D). In contrast, strains that overexpressed
Bro1 or just the Bro1 domain had reduced frequencies of
Rim20-GFP foci (Fig. 2B, C, and D). These results support the
model that Bro1 and Rim20 compete for association with en-
dosomes and that competition is mediated by the Bro1 do-
pH-independent endosomal localization of Rim20p requires
ESCRT machinery but not the RIM101 machinery. If Rim20
associates with ESCRT in the absence of Bro1, then ESCRT
subunits should be required for the novel Rim20-GFP foci that
form in the bro1? mutant background under acidic growth
conditions. Thus, we measured the accumulation of Rim20-
GFP foci in a panel of bro1? mutants lacking ESCRT subunits.
Mutations that eliminated ESCRT-I, -II and -III subunits abol-
ished most Rim20-GFP foci (Fig. 3A) compared to the WT
(Fig. 3C). Although foci were largely abolished under acidic
conditions (Fig. 3A and D), a few cells formed foci under
alkaline conditions (Fig. 3B). These results are consistent with
previous observations in BRO1 mutant strains (4). The depen-
dence of Rim20-GFP foci on ESCRT subunits argues that the
foci are endosomal compartments.
In addition to a shared requirement for ESCRT subunits,
localizations of Bro1 and Rim20 have distinct genetic require-
ments. If Rim20 enters the Bro1 localization pathway in the
bro1? mutant background, the formation of Rim20-GFP foci
in this background may have Bro1-specific genetic require-
ments. The VPS27 gene product promotes the association of
ESCRT-I with endosomes but is not required for Rim101
processing (4, 9, 25). Comparison of a vps27? bro1? mutant
strain to a bro1? mutant strain revealed comparable amounts
of Rim20 foci formed under alkaline conditions, when the
Rim101 pathway is active (Fig. 3B). However, fewer foci were
observed under acidic conditions in a vps27? bro1? mutant
strain than in a bro1? mutant strain (Fig. 3A and D). Analysis
of four Rim101 pathway genes (RIM8, RIM9, RIM21, DFG16),
positive regulators of alkaline pH-induced Rim20 localization,
showed that Rim20-GFP foci occurred in the bro1? mutant
background independently of these genes (Fig. 4). In addition,
loss of either the protease Rim13 or Rim101 itself had little
effect on Rim20-GFP foci in the bro1? mutant background
(Fig. 4A and D), whereas their loss causes increased numbers
FIG. 1. Acidic Rim20-GFP localization occurs in bro1? and vps4? mutant strains. Live-cell imaging of Rim20-GFP strains treated with acidic
medium (pH 4.0); WT control strain JBY46 (A), vps4? mutant strain JBY115 (B), and bro1? mutant strain JBY295 (C). (D) Quantification of
GFP foci in a population of 100 cells following acidic treatment.
534 BOYSEN ET AL.EUKARYOT. CELL
of Rim20-GFP foci in WT cells (4). We conclude that the novel
Rim20-GFP foci that arise in bro1? mutant strains have the
genetic requirements for Bro1-endosome association.
Association of Rim20 with endosomes is not sufficient for
Rim101 pathway activation. Previous studies in a vps4? mutant
background suggested that Rim20-endosome association al-
lowed some activation of Rim101 under otherwise restrictive
conditions. In vps4? mutant strains, there is considerable
Rim20-endosome association and partial Rim101 activation in
the absence of many Rim101 pathway members and ESCRT
subunits (4, 6). If endosomal Rim20 in a bro1? mutant back-
ground is sufficient for Rim101 processing, then mutant strains
that form Rim20-GFP foci should promote Rim101 activity, as
measured by repression of Rim101 target genes. Thus, we
examined the expression of Rim101 target genes RIM8 and
SMP1 under acidic and alkaline growth conditions. Rim101
represses both genes and is found associated with the promoter
sequences of both genes (11). As expected, expression of both
genes was low in the WT strain and elevated in a rim101?
mutant and in the Rim101 pathway mutant dfg16? deletion-
carrying strain (Fig. 5A and B). Loss of BRO1 did not restore
repression in the dfg16? mutant (Fig. 5A and B), despite the
presence of Rim20-GFP foci (Fig. 4). We conclude that asso-
ciation of Rim20 with endosomes due to lack of Bro1 is not
sufficient to promote Rim101 activity.
Rim20 localizes to endosomes under alkaline pH conditions
and is essential for proteolytic activation of the Rim101 tran-
scription factor. Prior studies with S. cerevisiae vps4? mutants
suggested that Rim20-endosome association may be the limit-
ing step for activation of Rim101 (4, 6). This model was based
on the finding that the vps4? defect promoted both Rim20-
endosome association and partial Rim101 activation under a
variety of conditions that were restrictive in VPS4 mutant
strains. We report here that a bro1? mutant strain separates
the two outcomes, permitting Rim20-endosome association
under otherwise restrictive conditions but not Rim101 activa-
tion. Our observations together indicate that there must be an
additional level of pH- and/or Rim101 pathway-dependent
control over the Rim20-Rim13-Rim101 complex that leads to
Our experiments suggest that Bro1 domain proteins, ex-
pressed at their natural levels, compete for endosome associ-
ation. These findings are consistent with the overall similarity
of the Bro1 domains of Bro1 and Rim20 and with the finding
that many alanine scan mutations in SNF7 impair both Bro1-
and Rim20-related functions (22). In fact, Rim20-Bro1 com-
petition may help to explain why snf7 mutants whose protein
products have only a twofold reduced ability to bind Bro1 in
FIG. 2. BRO1 overexpression inhibits alkaline Rim20-GFP focus formation. Live-cell imaging of Rim20-GFP strains treated with alkaline
medium (pH 8.3); WT control strain JBY46 (A), strain JBY46 carrying high-copy BRO11-367plasmid pJB31 (B), and strain JBY46 carrying
high-copy BRO1 plasmid pJB30 (C). (D) Quantification of Rim20-GFP foci observed in a population of 100 cells following alkaline treatment.
VOL. 9, 2010 Bro1 DOMAIN PROTEIN COMPETITION535
vitro have endocytic trafficking defects as severe as those of a
snf7? null mutant strain (21). Similarly, competition by Bro1
may contribute to the Rim101 pathway-specific phenotypes of
the alanine scan snf7 alleles (22). However, we did not see
functional consequences of such competition with WT Snf7 in
experiments reported here. It is likely that simple replacement
of Bro1 with Rim20 does not open up sites on the endosomes
for other essential upstream components of the Rim101 path-
way to assemble. In fact, it has been shown recently that Snf7
binding to both ESCRT proteins and Rim proteins is much
stronger under alkaline growth conditions (21), which might
explain why artificial Rim20 localization does not recapitulate
other changes in the cell upon a shift to alkaline pH. From
these results, it seems possible that the consequences of Bro1
domain competition may have been minimized through evolu-
Interestingly, when Rim20 substitutes for Bro1 at endosomal
sites, it assumes the regulatory requirements for Bro1-endo-
some association. Specifically, under acidic conditions in the
bro1? mutant background, Rim20-GFP foci are independent
of upstream Rim101 pathway components and become depen-
dent upon Vps27. This observation fits well with our mecha-
nistic understanding of ESCRT-I recruitment to endosomes.
Vps27 promotes ESCRT-I recruitment, presumably in re-
sponse to the monoubiquitination signal that marks plasma
membrane proteins for endocytosis (17, 23). Recent results
from Vincent and colleagues suggest that the Rim101 pathway
upstream components, via Rim8, may function analogously in
alkaline pH-dependent ESCRT-I recruitment (7). Thus, it
seems reasonable that most endosomal ESCRT under acidic
growth conditions depends upon Vps27, because Rim8 and
other upstream Rim101 pathway components are inactive.
Using double-mutant analysis in the absence of BRO1, we
find upregulation of Rim20 localization but no suppression of
Rim101 processing defects, which is unlike that seen in vps4
mutants. One idea to reconcile these findings is that trafficking
of Rim101 pathway components necessary for processing com-
plex formation may differ between the two mutant back-
grounds. This would imply that a trafficking defect exists in the
absence of Bro1 but not in the absence of Vps4, yet no traf-
ficking evidence supports this model. We favor a simpler ki-
netic model, based on a functional differentiation between
Vps4 and Bro1. Vps4 is an enzyme critical for endosomal
protein disassociation, while Bro1 is a structural protein whose
endosome disassociation is controlled by Vps4. In the absence
of Vps4, significant endosome-protein capture and sequestra-
tion occur, including Rim20 and Rim13—the protease that
cleaves Rim101—along with Rim101 itself and possibly other
components. In contrast, Bro1 is a structural protein, and loss
of Bro1 likely only affects the endosome association status of
Rim20, with little effect on any additional proteins. Consistent
with a carefully calibrated kinetic mechanism, as mentioned
above, Rim20 foci are most abundant immediately following
pH shock and recede within 10 min, suggesting an acute reg-
ulated time period of processing complex formation.
Previously, it was shown that in the absence of Bro1,
FIG. 3. ESCRT machinery is required for acidic Rim20-GFP localization. Live-cell imaging of Rim20-GFP double-mutant strains derived from
bro1? mutant strain JBY295 and treated with acidic (A) or alkaline (B) medium. (C) WT (JBY46) cells shifted to pH 4.0 or 8.3. (D) Quantification
of Rim20-GFP foci observed in a population of 100 cells following acidic treatment.
536 BOYSEN ET AL.EUKARYOT. CELL
Rim101 is processed efficiently but there is still a defect in
transcriptional repression by Rim101 (20). Repression was
assayed with a site that required the activity of both Rim101
and the repressor Nrg1. In our study, we quantitatively re-
corded the transcription of two native Rim101 targets, RIM8
and SMP1, and assayed RNA accumulation from the native
genomic loci. Both were repressed by Rim101 in WT and
bro1? mutant cells while being derepressed in dfg16? and
bro1? dfg16? mutant cells. Our findings indicate that a
bro1? mutation does not alleviate Rim101-dependent re-
pression at all target genes.
Our observations reported here argue that association of
Rim20 with endosomes has to be accompanied by an addi-
tional signal(s) mediated by alkaline pH that may change the
conformation of the ESCRT complex or its interaction with
Rim101 pathway components. This change mediated by a pH
shift may specialize some ESCRT-endosomes as hubs for
downstream signaling and open new sites to facilitate recruit-
ment of the protease Rim13 or other factors.
We are grateful to all members of our lab for helpful discussions and
for comments on the manuscript and to Miguel Pen ˜alva and members
of his lab for comments on this project.
This work was supported by National Institutes of Health grant
5R01AI070272 to A.P.M.
FIG. 4. Rim101 machinery is not required for acidic Rim20-GFP localization. Live-cell imaging of Rim20-GFP double-mutant strains derived
from bro1? mutant strain JBY295 and treated with acidic (A) or alkaline (B) medium. (C) Quantification of Rim20-GFP foci observed in a
population of 100 cells following acidic treatment.
FIG. 5. Loss of BRO1 does not suppress Rim101 processing de-
fects. qPCR analysis of Rim101 target genes RIM8 and SMP1 in WT
control and vps4?, rim101?, bro1?, dfg16?, and dfg16? bro1? mutant
(JBY324) cells after a 120-min shift in YPD plus 0.1 M HEPES at pH
4.0 (A) and pH 8.3 (B).
VOL. 9, 2010 Bro1 DOMAIN PROTEIN COMPETITION537
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