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From the
Received for publication, April 23, 2002, and in revised form, May 28, 2002
Yeast cells organize their actin cytoskeleton in
a highly polarized manner during vegetative growth. The Ras-like GTPase
Rsr1/Bud1 and its regulators are required for selection of a specific
site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud
site and polarized growth sites. We also showed that localization of
Cdc24, a guanine nucleotide exchange factor for the Cdc42
GTPase, to the proper bud site was dependent on Rsr1/Bud1.
Surprisingly, Rsr1/Bud1 also localized to intracellular membranes. A
mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a
mutation at the CAAX motif eliminated both plasma
membrane and internal membrane association of Rsr1/Bud1. Thus the
lysine repeat and the CAAX motif of Rsr1/Bud1 are important
for its localization to the plasma membrane and to the polarized growth
sites. This localization of Rsr1/Bud1 is essential for its function in
proper bud site selection because both mutations resulted in random bud site selection.
Cells of the budding yeast Saccharomyces cerevisiae
undergo oriented cell division by selecting a specific site for
polarized growth on their cell cortex (1-3). Haploid a and
To understand the mechanism of action of the Rsr1 GTPase module, it is
crucial to determine whether any of its components are localized to the
presumptive bud site. We reported previously that both Bud2 and Bud5
are localized to the presumptive bud site and to discrete sites
during the cell cycle (10, 11). This localization is essential
for selection of a specific site for growth. Here we report that Rsr1
is broadly distributed on the plasma membrane and is highly
concentrated at the incipient bud site and polarized growth sites.
Mutational studies indicated that the lysine repeat in the
hypervariable region and the CAAX motif of Rsr1 are
important for its localization and its role in selection of a proper
site for growth. We also show that localization of Cdc24, a guanine
nucleotide exchange factor for Cdc42, to the proper bud site is
dependent on Rsr1.
Plasmids and Yeast Strains--
Yeast genetic manipulations were
performed as described previously (12). Yeast strains used in
this study are listed in Supplemental Table I. Cdc24-green fluorescent
protein (GFP)1 was expressed
using plasmid pYS47 (a gift from M. Peter) (13). YEp13-rsr1G12V and YEp13-rsr1K16N were gifts
from M. Ruggieri (14). Plasmids expressing the wild-type and mutant
rsr1 as GFP or yellow fluorescent protein (YFP) fusions are
described below.
Construction of Strains Expressing GFP-Rsr1--
To express Rsr1
fused to GFP, first a NotI site was introduced right after
the start codon of RSR1 in PB290 (a gift from A. Bender) (4)
by site-directed mutagenesis using the QuikChange site-directed
mutagenesis kit (Stratagene), resulting in pHP764. A 720-bp
NotI fragment coding for GFP(S65T,V163A,S175G) was
amplified by PCR from pAFS144 (a gift from A. F. Straight) (15)
and cloned into the NotI site of pHP764, resulting in
plasmid pHP767. To construct an integrating plasmid, the
SacI-SalI fragment of pHP767 was cloned into
pRS304, resulting in pHP808. A haploid Construction of Strains Expressing YFP-Rsr1G12V and
YFP-Rsr1K16N--
The rsr1G12V and
rsr1K16N alleles were generated by PCR-based
site-directed mutagenesis using pHP818 as a template. Mutations were confirmed by DNA sequencing. The resulting plasmids, pHP843 and pHP844,
were integrated into the RSR1 locus of an Construction of Strains Expressing GFP-Rsr1C269S and
GFP-Rsr1K260-264S--
The
rsr1C269S mutation was introduced by PCR-based
site-directed mutagenesis using pHP808 as a template, resulting
in pHP1042. The rsr1K260-264S mutation was
generated by PCR-based mutagenesis in two steps. First,
rsr1K263-264S was generated using pHP808 as a
template. The resulting plasmid, pHP1041, was then used as a template
to generate the rsr1K260-264S mutation,
resulting in pHP1065. The plasmids pHP1042 and pHP1065 were integrated
into the RSR1 locus of HPY263, resulting in HPY590 and
HPY621, respectively.
Microscopy--
To view GFP-Rsr1 or YFP-Rsr1, cells were grown
to early log phase and visualized using a Nikon E800 microscope fitted
with a 100× immersion objective (N.A. = 1.30) as described previously (10) using filters from Chroma (Brattleboro, VT). Images were collected
using a Micromax digital camera (Princeton Instruments) and IPLab
software (Signal Analytics Corp., Vienna, VA). To collect images of
several Z-sections, motorized stage movement was driven using
PerkinElmer Life Sciences Imaging Suite software (version 4.1). Bud
scars and birth scars were visualized by staining cells with Calcofluor
as described previously (16). The vacuole lumen was visualized using
CellTracker Blue CMAC (Molecular Probes) as suggested in the
manufacturer's protocol. To stain DNA in living cells expressing GFP,
cells in growth medium were incubated with DAPI (at 10 µg/ml) at room
temperature for 5 min and were washed once with growth medium before observation.
Rsr1 Is Enriched at the Incipient Bud Site and the Polarized Growth
Site--
The localization of Rsr1 in haploid yeast cells was
determined using a chromosomal GFP-RSR1 fusion
that complemented an rsr1 deletion (Supplemental Table II).
Although GFP-Rsr1 appeared to be broadly distributed on the plasma
membrane throughout the cell cycle in most cells, enrichment of
GFP-Rsr1 at the sites of polarized growth was notable (Fig.
1A, see also Fig.
3B). GFP-Rsr1 appeared to be evenly distributed on the
plasma membrane in over 50% of unbudded cells (n = 145, 1) and was enriched in a patch at the incipient bud
site (20%; Fig. 1A, 2, 3, and
8). After bud emergence, GFP-Rsr1 was enriched at the
periphery of growing buds (89% of cells with small- and medium-sized
buds, n = 70; Fig. 1A, 4 and 5) and at the mother/bud neck at a later stage of cell cycle
(55% of cells with large-sized buds, n = 100; Fig.
1A, 6). Upon cytokinesis and cell separation,
GFP-Rsr1 appeared to be more concentrated at a small portion of the
plasma membrane, which corresponds to the division site, than in the
rest of the plasma membrane (26% of G1 cells; Fig.
1A, 7), and a new patch of GFP-Rsr1 appeared again before bud emergence (Fig. 1A, 8).
Localization of GFP-Rsr1 in diploid a/
We also noticed intracellular distribution of GFP-Rsr1 in addition to
localization to the plasma membrane and sites of polarized growth (see
below). To confirm that the intracellular distribution was not
background signal of a GFP fusion, we also expressed RSR1 as
a YFP fusion protein from its own chromosomal locus, which also
complemented an rsr1 deletion (Supplemental Table II). The localization pattern of YFP-Rsr1 was indistinguishable from that of
GFP-Rsr1 (Fig. 1B), although overall signal was slightly weaker.
Since Rsr1 can exist in GTP- and GDP-bound states, we wished to know
whether Rsr1 localized differently depending on its guanine nucleotide-bound state. Thus we expressed the rsr1 mutants,
which would be locked in a GTP- or GDP-bound state in vivo,
and examined their localization as YFP fusion proteins. Each YFP-rsr1
mutant was expressed from the RSR1 chromosomal locus, and
the level of the protein was approximately equal to the wild-type
protein (data not shown). The apparent localization pattern of
YFP-Rsr1K16N, which is predicted to be a GDP-bound or
nucleotide-empty state in vivo (14), was not significantly
different from that of wild-type YFP-Rsr1, but some notable differences
were found (Fig. 1B). Localization of
YFP-Rsr1K16N to the polarized growth sites in cells with
small- or medium-sized buds was diminished (30%, n = 70) compared with that of the wild-type cells (89%). The signal at the
mother/bud neck appeared less tightly organized and often only at the
mother or bud side (Fig. 1B, arrow). In contrast,
YFP-Rsr1G12V, which is predicted to be a constitutively
GTP-bound state (14), showed a more dramatic difference compared with
the wild-type cells: the overall YFP signal on the plasma membrane and
internal membranes was greatly enhanced uniformly.
YFP-Rsr1G12V failed to be concentrated at the sites of
polarized growth: less than 5% of unbudded cells (n = 100) showed enriched signals at the incipient bud site. Less than 10%
of cells with small- or medium-sized buds (n = 70) and
20% of cells with large-sized buds (n = 120) showed
polarized localization of YFP-Rsr1G12V (Fig.
1B). Since both rsr1G12V and
rsr1K16N mutations lead to random bud site
selection (14), these results, together with data discussed below,
suggest that localization of Rsr1 to the plasma membrane is necessary
but not sufficient for its function. Its enrichment at the incipient
bud site and polarized growth sites and continuous cycling of Rsr1-GTP
and Rsr1-GDP are likely to be important for proper bud site selection.
Rsr1 Also Localizes to the Vacuole Surface--
As shown above,
GFP-Rsr1 localized to internal membranes in addition to the polarized
growth sites and the plasma membrane. To determine whether GFP-Rsr1
localizes to any internal organelle specifically, cells expressing
GFP-Rsr1 were stained with a dye that stains either DNA or the vacuole
lumen. GFP-Rsr1 rarely localized to the boundary of nucleus stained
with DAPI; rather it often appeared as a circular ring that did not
overlap with the nucleus (Fig. 2). Next
we stained cells with CellTracker Blue CMAC, a dye that stains the
vacuole lumen. In most cells (>90%), the GFP-Rsr1 ring appeared right
outside of the blue staining, indicating that GFP-Rsr1 localized to the
vacuole surface (Fig. 2).
It has been reported that overexpression of many membrane proteins that
function elsewhere, such as the Golgi apparatus or plasma membrane, can
cause accumulation of the protein within the vacuole (17). However,
these proteins were mainly targeted to the lumen of vacuole rather than
the surface. This phenomenon is most apparent in cells lacking the
vacuole proteases that allow these proteins to accumulate without
degradation (17, 18). Thus we examined GFP-Rsr1 localization in a
pep4 mutant that lacked vacuole proteases to ensure that
proteins localized to the lumen of the vacuole could be visualized.
GFP-Rsr1 localized to the vacuole surface in pep4 mutant
cells in a manner similar to its localization in PEP4
cells (data not shown), indicating that GFP-Rsr1 does not localize
within the interior of the vacuole.
The CAAX Motif and the Lysine Repeat in the Hypervariable Region of
Rsr1 Are Required for Its Localization and for Its Role in Bud Site
Selection--
It has been reported recently that intracellular
membranes and vesicular transport are involved in the passage of Ras
proteins to the plasma membrane (19). The signal required for Ras to be
localized to the plasma membrane consists of two components: the
C-terminal CAAX motif and either palmitoylation sites or a polybasic stretch of amino acids in the hypervariable region near the C
terminus (19). To confirm that the CAAX motif is necessary for membrane targeting of Rsr1, we expressed
rsr1C269S (cysteine to serine change in the
CAAX motif, Fig.
3A) as a GFP fusion from the
RSR1 locus on the chromosome. This mutation resulted in
random bud site selection (Supplemental Table II) as expected. Collecting images of several Z-sections by motorized stage movement of
the microscope, we confirmed that GFP-Rsr1C269S was
distributed homogeneously in the cytoplasm (Fig. 3B). These results suggest that the intact CAAX motif is required for
localization of Rsr1 to the plasma membrane and intracellular
membranes.
Rsr1 also contains five contiguous lysine residues (amino acids
260-264) in the hypervariable region (Fig. 3A), all of
which we changed to serine. Expression of
GFP-Rsr1K260-264S from the chromosome resulted in
completely random budding unlike that seen with the wild-type Rsr1
fused to GFP (Supplemental Table II). Thus the lysine residues are
essential for the function of Rsr1 in bud site selection.
Interestingly, GFP-Rsr1K260-264S still localized to
internal membranes, but it no longer localized to polarized growth
sites and to the plasma membrane (Fig. 3B). Both
GFP-rsr1C269S and
GFP-rsr1K260-264S were expressed at about the
same level as the wild type (data not shown), indicating that the
difference in localization pattern of Rsr1 and its mutants was not due
to a different level of each protein. Taken together, these data
suggest that the CAAX motif and the lysine repeat in the
hypervariable region of Rsr1 are required for localization to the
plasma membrane and polarized growth sites, which is essential for
proper bud site selection.
Rsr1 Is Necessary for Localization of Cdc24-GFP to the Proper Bud
Site in Late G1 Phase--
We proposed previously that the
Rsr1 GTPase cycle functions to guide proteins necessary for bud
formation, such as Cdc24, to the proper bud site (8). It has been
recently reported that Cdc24 localizes to the nucleus in early
G1 cells through the association with Far1 and to an
incipient bud site in late G1 phase (13, 20, 21). However,
it is not known how Cdc24 localizes to the incipient bud site in late
G1 phase. We postulated that localization of Cdc24 to the
proper bud site is dependent on Rsr1. Thus we examined the localization
of Cdc24-GFP in the wild-type and rsr1
It has been shown previously that Cdc24 specifically interacts with the
GTP-bound Rsr1 in vitro (8, 22). Thus we also examined
localization of Cdc24-GFP in cells carrying
rsr1G12V or rsr1K16N on a
multicopy plasmid. Localization of Cdc24-GFP in cells carrying YEp-rsr1K16N was not significantly different from that of
cells carrying the vector control except that a patch of Cdc24-GFP
signal in unbudded cells appeared at a random bud site (Fig.
4B). In contrast, localization of Cdc24-GFP in cells
carrying YEp-rsr1G12V was greatly altered compared with
that of cells carrying the vector control (Fig. 4B):
Cdc24-GFP was broadly distributed on the plasma membrane and
intracellular membranes in unbudded cells (52%, n = 390) and sometimes in more than one patch. Cells with enriched signal
of Cdc24-GFP at the periphery of growing buds also showed increased GFP
signal on the plasma membrane and internal membranes (Fig.
4B). Mislocalization of Cdc24-GFP was also observed in cells
carrying the wild-type RSR1 on a multicopy plasmid but to a
much less extent (data not shown). These results suggest that
overexpression of a GTP-locked form of Rsr1 recruits Cdc24 uniformly to
the plasma membrane.
We showed previously that the regulators of Rsr1, Bud2 and Bud5,
are localized to the presumptive bud site and to discrete sites
during the cell cycle and that their localization is essential for selection of a specific site for growth (10, 11). Bud2 and Bud5
cannot maintain their localization at the proper bud site in
rsr1 We also show that localization of Cdc24 to the proper bud site in the
G1 phase is dependent on Rsr1. In the absence of Rsr1, Cdc24 localized to a random location relative to the previous division site. This Cdc24 localization to a random site may
occur through a distinct default pathway yet to be identified or by the
stochastic accumulation of bud site assembly proteins on the plasma
membrane. Moreover, we found that Cdc24 was mislocalized in cells
overexpressing an rsr1 mutant that is predicted to be constitutively in the GTP-bound state in vivo. These data
indicate that Cdc24 is targeted to the proper bud site through the
interaction with Rsr1-GTP and that cycling of Rsr1 between GTP- and
GDP-bound states is required for targeting of Cdc24 to the proper bud
site, consistent with previous reports (8, 14). Thus, these data further support the role of the Rsr1 GTPase cycle in guiding the bud
site assembly proteins to the proper bud site (8).
Interestingly, cells can still polarize their growth to form a bud,
although in a random place, even when Cdc24 is mislocalized in cells
carrying YEp-rsr1G12V. Uniform localization of Cdc24 to the
plasma membrane due to overexpression of Rsr1G12V did not
cause a multibudding phenotype, although rounder and larger cells were
found (Fig. 4B). It is possible that the uniform localization of Cdc24 to the plasma membrane may not be sufficient to
cause activation of Cdc42, whereas enrichment of Cdc24 in a patch can
activate Cdc42. Alternatively, Cdc24 that is distributed uniformly
around the plasma membrane in cells overexpressing Rsr1G12V
may not be active because GTP-locked Rsr1 sequesters Cdc24 in an
inactive state as proposed previously (8).
Surprisingly, we found that Rsr1 also localized to the surface
of vacuole. Localization to the vacuole does not necessarily indicate
that Rsr1 functions at the vacuole and may result from a transient
association of Rsr1 with intracellular membranes during its trafficking
to the plasma membrane as seen with mammalian Ras proteins (19).
However, it is interesting to note that several recent reports suggest
a link between the Cdc42 signaling pathway and vacuolar function and/or
morphology (24-27). Thus the Rsr1 GTPase, which is likely to function
upstream of Cdc42 (3, 8), may also be involved in vacuolar function.
We thank A. Simcox and E. Angerman for
comments on the manuscript; A. F. Straight, T. Davis, M. Ruggieri,
A. Bender, and M. Peter for plasmids; and D. Hailey, B. Glick, D. S. Goldfarb, L. S. Weismann, and T. Richman for technical
suggestions. We also thank S. A. Osmani and C. P. C. De
Souza for help and for use of the microscope.
*
This work was supported by National Institutes of Health
Grant R01 GM56997 (to H.-O. P.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Molecular
Genetics, The Ohio State University, 484 West 12th Ave., Columbus, OH
43210-1292. Tel.: 614-688-4575; Fax: 614-292-4466; E-mail: park.294@osu.edu.
Published, JBC Papers in Press, June 10, 2002, DOI 10.1074/jbc.C200245200
The abbreviations used are:
GFP, green
fluorescent protein;
YFP, yellow fluorescent protein;
DAPI, 4',6-diamidino-2-phenylindole dihydrochloride;
CMAC, 7-amino-4-chloromethylcoumarin.
ACCELERATED PUBLICATION
Localization of the Rsr1/Bud1 GTPase Involved in Selection of a
Proper Growth Site in Yeast*,
§¶,, and Department of Molecular Genetics and
§ Graduate Program in Molecular, Cellular, and Developmental
Biology, The Ohio State University, Columbus, Ohio 43210-1292
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells bud in an axial pattern, whereas diploid
a/ cells bud in a bipolar pattern. The GTPase module
consisting of Rsr1/Bud1 (Rsr1 hereafter), its GDP-GTP exchange factor
Bud5, and its GTPase-activating protein Bud2 is essential for
selecting the proper site for polarized growth in both haploid and
diploid cells (4-7). Based on genetic and biochemical data, we
proposed previously that the Rsr1 GTPase module directs bud site
assembly to occur at specific locations by recruiting components such
as Cdc24 required for bud formation to that site (8). Recruiting these
proteins to the presumptive bud site is thought to direct the
cytoskeleton and secretory apparatus toward the bud site, thereby
restricting new growth to the bud (9). One of the key questions in
understanding the molecular basis of cell polarity is how specific
sites for actin polymerization are determined.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain expressing GFP-Rsr1
(HPY401) was constructed by integration of pHP808 digested with
BssHII into the RSR1 locus of an rsr1
strain (HPY263). A diploid a/ strain
expressing GFP-Rsr1 (HPY618) was constructed using strain HPY401 and
the plasmid pRS315-HO. Similarly, a plasmid expressing YFP-Rsr1
(pHP818) was generated by PCR using the plasmid pDH5 as a template (a
gift from Yeast Biology Resource Center, University of
Washington, Seattle, WA).
rsr1 strain (HPY263), resulting in HPY422 and HPY423, respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells was very
similar to that in haploid a or cells (data not
shown).
View larger version (55K):
[in a new window]
Fig. 1.
Localization of GFP-Rsr1 or YFP-Rsr1 in
haploid cells. A, localization of GFP-Rsr1 in haploid
cells. Representative images of yeast cells expressing GFP-Rsr1
(strain HPY401) are shown. Panel 8 shows two cells after
cytokinesis and cell separation. B, localization of
YFP-Rsr1, YFP-Rsr1K16N, and YFP-Rsr1G12V in
haploid cells. Representative images of yeast cells expressing
YFP-Rsr1 (strain HPY402), YFP-Rsr1G12V (strain HPY422), or
YFP-Rsr1K16N (strain HPY423) are shown.
View larger version (47K):
[in a new window]
Fig. 2.
Localization of GFP-Rsr1 to intracellular
membrane. Nucleus or vacuolar lumen was localized by staining
cells (strain HPY401) with DAPI or CellTracker Blue CMAC,
respectively.
View larger version (62K):
[in a new window]
Fig. 3.
The CAAX motif and the
polylysine repeat of Rsr1 are necessary for its proper
localization. A, the hypervariable region of Rsr1 with
the CAAX motif underlined. The residues mutated
in rsr1C269S and
rsr1K260-264S are indicated. B,
localization of GFP-Rsr1C269S,
GFP-Rsr1K260-264S, and GFP-Rsr1 wild type.
Typically 10 Z-sections of cells expressing the GFP-Rsr1 (strain
HPY401), GFP-Rsr1C269S (strain HPY590), or
GFP-Rsr1K260-264S (strain HPY621) were examined by
motorized stage movement of the microscope. A representative section is
shown for each strain.
cells that were
also stained with Calcofluor to visualize the previous division sites.
As shown in Fig. 4A, Cdc24-GFP
localized to a site adjacent to the previous division site in wild-type cells. In contrast, Cdc24-GFP localized to a random site in
rsr1 cells, indicating that localization of Cdc24 to the
proper bud site is dependent on Rsr1.
View larger version (37K):
[in a new window]
Fig. 4.
Localization of Cdc24-GFP to the proper bud
site is dependent on Rsr1. A, localization of Cdc24-GFP
in the wild-type or rsr1 strains. Representative images
of Cdc24-GFP (green) and Calcofluor staining
(blue) are shown for the wild-type (strain IH1784) and
rsr1 (strain HPY263) cells carrying YCp-CDC24GFP.
Arrows indicate the position where Cdc24-GFP localizes in
unbudded cells. The arrowhead indicates a birth scar marking
the division site in a daughter cell. B, localization of
Cdc24-GFP in cells carrying YEp13, YEp-rsr1K16N, or
YEp-rsr1G12V. Approximately 100 cells (strain IH1783) were
examined for each panel (except for YEp-rsr1G12V for which
390 unbudded cells were examined), and representative images are
shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells. Here we report that Rsr1 was also enriched at
the incipient bud site and polarized growth sites. Expression of
Rsr1G12V or Rsr1K16N, which causes a random bud
site selection (14), resulted in much less polarized localization. In
particular, Rsr1G12V, which is predicted to be a GTP-locked
state in vivo (14), localized uniformly to the plasma
membrane. Similarly, we found that localization of Rsr1 to the
incipient bud site and polarized growth sites was diminished in a
bud2 mutant but to a lesser extent compared with that of
Rsr1G12V (data not shown). Thus these studies indicate that
localization of Rsr1 to the incipient bud site and polarized growth
sites is important for proper bud site selection. Our findings support the view that selection of a proper site for growth requires the localized action of all components of the Rsr1 GTPase module. It has
been reported previously by indirect immunofluorescence in a strain
carrying RSR1 on a multicopy plasmid that Rsr1 localizes uniformly around the plasma membrane (23). It is likely that the
previous study by Michelitch and Chant (23) missed enrichment of Rsr1
at the sites of polarized growth and on internal membranes due to
overexpression of Rsr1 or fixation of yeast cells for immunofluorescence.
ACKNOWLEDGEMENTS
FOOTNOTES
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Tables I and II.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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