ArabidopsisSorting inhibitors (Sortins): Chemical compounds to study vacuolar sorting in
Jan Zouhar, Glenn R. Hicks, and Natasha V. Raikhel
doi:10.1073/pnas.0402121101
2004;101;9497-9501; originally published online Jun 9, 2004;PNAS
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Sorting inhibitors (Sortins): Chemical compounds
to study vacuolar sorting in Arabidopsis
Jan Zouhar, Glenn R. Hicks, and Natasha V. Raikhel*
Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521
Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved May 5, 2004 (received for review March 25, 2004)
Chemical genomics is an interdisciplinary approach that unites the
power of chemical screens and genomics strategies to dissect
biological processes such as endomembrane trafficking. We have
taken advantage of the evolutionary conservation between plants
and Saccharomyces cerevisiae to identify such chemicals. Using S.
cerevisiae, we screened a library of diverse chemical structures for
compounds that induce the secretion of carboxypeptidase Y, which
is normally targeted to the vacuole. Among 4,800 chemicals
screened, 14 compounds, termed sorting inhibitors (Sortins), were
identified that stimulated secretion in yeast. In Arabidopsis seedlings,
application of Sortin1 and -2 led to reversible defects in
vacuole biogenesis and root development. Sortin1 was found to
redirect the vacuolar destination of plant carboxypeptidase Y and
other proteins in Arabidopsis suspension cells and cause these
proteins to be secreted. Sortin1 treatment of whole Arabidopsis
seedlings also resulted in carboxypeptidase Y secretion, indicating
that the drug has a similar mode of action in cells and intact plants.
We have demonstrated that screening of a simple eukaryote, in
which vacuolar biogenesis is not essential, can be a powerful tool
to find chemicals that interfere with vacuolar delivery of proteins
in plants, where vacuole biogenesis is essential. Our studies were
done by using a sublethal dose of Sortin1, demonstrating the
powerful ability of the chemical to control the induced phenotype
in a manner that would be difficult to achieve using conventional
genetics.
In plant biology, the isolation of T-DNA [portion of the Ti
(tumor-inducing) plasmid that is transferred to plant cells]
gene inactivation mutants has become a valuable tool for
understanding gene function. However, the availability of viable
and informative knockout mutants in genes involved in protein
endomembrane trafficking is still limited. This constraint is due
to the fact that many genes involved in trafficking have proven
to be essential for gametophyte or embryo development (1­3)
whereas others are in gene families containing members that are
redundant functionally and can fully or partially relieve mutation
effects (4). Although a method has been developed for the
isolation of point mutants that may overcome the lethality
associated with insertional gene knockouts (5), the development
of additional tools that address the function of essential or
redundant gene products would be extremely beneficial. Furthermore,
plant biology would profit enormously from approaches
that would yield both reversible and tunable plant
responses, which are difficult or impossible to achieve using
conventional genetic approaches.
Chemical genomics (genomics scale chemical genetics) offers
such a powerful tool. This approach involves the screening of
collections of synthetic compounds for those having desirable
biological activities. The cognate targets of such compounds may
identify novel pathways or novel interactions with known pathways.
The cognate targets of biologically active compounds can
then be determined by using the advanced genetics in plant
systems such as Arabidopsis thaliana (6). This approach is in some
ways analogous to that of classical forward genetics, in which
collections of mutants are used to dissect pathways (for review,
see refs. 7­10), except that chemical genomics offers distinct
advantages. Because chemical libraries can be stored and
screened in ordered arrays, one tremendous advantage is the
ability to perform screening assays in a high-throughput or even
automated mode. The design of such high-throughput screens
that focus specifically on particular subcellular pathways may
also pose challenges for plant biologists because plants are
multicellular organisms with a highly complex developmental
cycle. Nevertheless, for some pathways, in particular those that
are evolutionarily conserved and cell autonomous, it may be
possible to take advantage of simpler single-celled eukaryotes
such as yeast. Although there are clear differences in protein
endomembrane trafficking in plants and yeast (11), such as the
fact that vacuoles are essential in plants but dispensable in yeast
(3, 12), the machineries share similarities such as homologous
genes and protein complexes (13, 14). Therefore, a potentially
valuable approach to identify drugs that affect the endomembrane
system of plants is to perform chemical screening employing
Saccharomyces cerevisiae. In our assay, we focused on the
identification of compounds that would alter the delivery of
carboxypeptidase Y through the endomembrane system into the
vacuole. Among the compounds identified, several drugs termed
Sortins were biologically active in Arabidopsis plants and suspension
cultures. Our results clearly demonstrate the power of
this approach for identifying novel plant-active compounds.
More importantly, we have discovered drugs to study the endomembrane
system of plants, which has proven challenging to
dissect by conventional genetics.
Methods
Phenotype Assays. S. cerevisiae INVSc1 (his3- 1, leu2, trp1-289,
ura3-52; Invitrogen) was used as a wild-type strain throughout
this study and does not display secretion of carboxypeptidase Y
(CPY) under normal conditions. A culture was grown in yeastpeptone-dextrose
(YPD) medium at 28°C with constant shaking
for 24 h. Yeast cells were then diluted 1,000 times in fresh media
and plated in V-shaped 96-well polypropylene plates (Greiner
Bio-One, Longwood, FL) at 100 l per well, supplemented with
chemical compounds in DMSO (4,800 from the DIVERSetE,
Chembridge, San Diego) at a concentration of 10 mg liter.
Non-drug control cells were treated with DMSO only. After 2
days of incubation at 28°C with constant shaking, cells were
centrifuged at 2,000 g, and 60 l of the growth media was
transferred onto a nitrocellulose membrane by using a dot-blot
apparatus (Bio-Rad). The membrane was then washed briefly
with water and blocked with 5% milk in PBS. Secreted CPY was
detected by monoclonal anti-CPY antibodies (Molecular
Probes). The yeast vacuolar morphology was subsequently visualized
by using a Leica TCS SP2 UV Confocal Microscope
(Leica Microsystems, Wetzlar, Germany) and the dye MDY-64
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Sortin, sorting inhibitor; CPY, carboxypeptidase Y; AtCPY, Arabidopsis
homologue of CPY; EGFP, enhanced GFP; TIP, tonoplast intrinsic protein; ER, endoplasmic
reticulum.
*To whom correspondence should be addressed at: Department of Botany and Plant
Sciences and Center for Plant Cell Biology, 2109 Batchelor Hall, University of California,
Riverside, CA 92521. E-mail: natasha.raikhel@ucr.edu.
 2004 by The National Academy of Sciences of the USA
www.pnas.org cgi doi 10.1073 pnas.0402121101 PNAS June 22, 2004 vol. 101 no. 25 9497­9501
PLANTBIOLOGY
(Molecular Probes). A vps16 deletion strain was used as a
control yeast strain with altered vacuolar morphology (12).
Arabidopsis seedlings expressing enhanced GFP (EGFP): tonoplast
intrinsic protein (TIP) chimeric protein (15) were
germinated on Murashige and Skoog agar in the presence of
chemicals. Vacuolar morphology was analyzed as described in
ref. 16. To investigate the effect of the compounds at the
molecular level, Arabidopsis and tobacco BY2 suspension cells
(cultured as described in refs. 17 and 18) were exposed to drugs
at various concentrations as indicated. Plant cell viability was
visualized with fluorescein-diacetate (FDA) (19). In live, metabolically
active cells, the nonfluorescent FDA is cleaved by
esterases into a fluorescent product, whereas inactive, dead cells
remain nonfluorescent (20).
Identification of Secreted Proteins and Pulse­Chase Experiments. To
identify proteins that might be secreted after a chemical treatment,
the growth medium was collected, concentrated, and
analyzed by SDS PAGE and immunoblotting. In addition, a
pulse­chase experiment (21) was done to examine the secretion
of an Arabidopsis homologue of CPY [AtCPY (22)] in the
presence of Sortin1. Briefly, 1.2 ml of 4- to 5-day-old cells were
distributed into 12-well microtiter plates and incubated with 132
Ci (1 Ci 37 GBq) of Expre35
S35
S label (Perkin­Elmer) on an
orbital shaker at 100 rpm. After 15 h, labeled proteins were
chased by adding unlabeled methionine and cysteine to a
concentration of 5 mM and 2.5 mM per well, respectively, and
the cells were supplemented with either drug in DMSO (at 25
mg liter, 57 M) or DMSO (control). At the time points
indicated, cells were pelleted by centrifugation for 30 s at
15,000 g. The growth media were then subjected to immunoprecipitation
as described in refs. 23 and 24 by using anti-AtCPY
antibodies (22) and fractionated by SDS PAGE, treated with a
scintillant (Fluoro-Hance, Research Product International,
Mount Prospect, IL), and fluorographed for 10 days.
Immunoelectron Microscopy. Arabidopsis seedlings were germinated
in the presence of 57 M Sortin1. Subsequently, sections
of hypocotyls and roots were prepared (22) and used for all
immunogold-labeling experiments as described in ref. 25. Controls
were performed with the use of the corresponding preimmune
serum substituted for the antisera. In all cases, the antisera
demonstrated high specificity of the labeling.
Results
Screening for Biologically Active Compounds in Yeast. In wild-type
yeast strains, CPY is delivered to the vacuole by means of
Golgi-to-endosome and endosome-to-vacuole pathways and
cannot be detected outside the cell. Yeast vacuolar protein sorting
(vps) mutants, by contrast, secrete a significant amount of CPY
(26). This vps phenotype resulting from treatment with specific
chemicals was the basis of our screen. We screened 4,800
compounds using an anti-CPY dot-blot immunoassay for the
CPY secretion phenotype in the yeast strain INVSc1, which does
not secrete CPY under normal conditions. The library chosen
was available commercially and was composed of a diverse set of
chemical structures (www.hit2lead.com). The library was supplied
in a mg liter format and has been used successfully by other
researchers (27). As predicted for the screening of a library of
diverse chemical structures, the preponderance of compounds
(99%) had no detectable effect in initial screens. Nine compounds
had fungicidal properties resulting in yeast lethality and
were not examined further (data not shown). Forty compounds
(0.8% of the total) were identified as ``hits'' in the primary
screen. We confirmed that 14 (0.3% of the total) of these 40
compounds possessed biological activity at concentrations of 10
mg liter and 100 mg liter. The identification numbers of these
14 compounds according to the Chembridge database
(www.hit2lead.com) and a qualitative description of the resulting
CPY secretion under the primary screening concentration are
presented in Table 1.
Based on the primary screen phenotype, we named these
drugs ``Sortins'' for protein sorting inhibitors. Two of them,
Sortin1 (Fig. 1A) and Sortin2 (Fig. 1B) were considered highpriority
hits based on the significant amount of secreted CPY
(Fig. 1 D and E). Interestingly, doses of Sortin1 25 mg liter
resulted in lesser, although still significant, stimulation of secretion.
Although we do not understand the basis of this observation,
the experiment was highly reproducible. The remaining 12
Table 1. Effects of Sortins on the CPY secretion of yeast (vps
phenotype) or the morphology of Arabidopsis or yeast vacuoles
Chembridge ID no. vps phenotype Vacuolar morphology
6168516 (Sortin1) Strong Arabidopsis
6239069 (Sortin2) Strong Arabidopsis
6108321 (Sortin3) Weak Yeast
5938112 Weak --
6129791 Weak --
6142844 Weak --
6150489 Weak --
6155208 Weak --
6169464 Weak --
5729136 Weak --
6124558 Weak --
6125186 Weak --
6127923 Weak --
6240429 Weak --
Strong secretion is typified by Sortin1 and -2 (see Fig. 1 D and E), whereas
weak secretion is typified by Sortin3 (see Fig. 1F).
Fig. 1. Sortins trigger CPY secretion in yeast. Shown are chemical structures
of Sortin1 (A, MW 441.44), Sortin2 (B, MW 429.92), and Sortin3 (C, MW 391.32).
A dot-blot assay indicated that Sortin1 (D) and Sortin2 (E) generated strong
vps phenotypes. In addition to a weak vps phenotype (F), Sortin3 induced a
severe vam phenotype (H), similar to that of Class C vps yeast mutant vps16
(I). The tonoplast morphology of untreated yeast is shown (G). Concentrations
are as indicated. (Scale bars 5 m.)
9498 www.pnas.org cgi doi 10.1073 pnas.0402121101 Zouhar et al.
compounds showed a weak degree of secretion enhancement
compared with Sortin1 and -2, which was typified by Sortin3 (Fig.
1F). Untreated control cells showed little or no detectable
secretion using the immunoblot assay (Fig. 1 D­F; 0 mg liter).
In addition to the vps phenotype, yeast cells were examined for
an altered vacuolar morphology (vam) phenotype (28). When
viewed by confocal microscopy using the specific vacuolar membrane
dye MDY-64, 13 of the 14 compounds resulted in no
obvious effect on yeast vacuolar morphology (Table 1). However,
one of the compounds, Sortin3 (Fig. 1C), when compared
with the untreated control cells (Fig. 1G), induced a dramatic
and severe vam phenotype in yeast (Fig. 1H). This phenotype
was similar to that of the control yeast mutant vps16 , which
displays a vam phenotype (12) (Fig. 1I). In a search of the
SciFinder database (Version 2004, American Chemical Society),
we found no references to compounds with the structures of
Sortin1, -2, and -3 having biological activity.
Sortins Result in Aberrant Vacuoles in Intact Plants and Are Reversible.
All fourteen compounds were tested on Arabidopsis plants
expressing EGFP: -TIP (15). Seedlings were germinated and
grown in the presence of the drugs for 1 week and examined for
vacuolar phenotype by confocal microscopy. Interestingly, compared
with untreated control plants (Fig. 2A), the vacuolar
membranes in hypocotyls of seedlings grown in the presence of
100 mg liter (227 M) Sortin1 or Sortin2 (233 M), highpriority
hits from the initial screen, seemed highly fragmented
(Fig. 2 B and C). These findings suggest that the compounds
likely target the vacuolar biogenesis machinery in plants. The
remaining 12 compounds had no effect on vacuolar morphology
at 100 mg liter tested (data not shown).
In addition to a tonoplast morphology phenotype, Sortin1 also
severely affected root development at 100 mg liter. Despite this
dramatic effect, the phenotype induced by Sortin1 was found to
be reversible after transfer to non-drug medium (Fig. 2D).
Sortin2 treatment also resulted in a dramatic inhibition of root
development at 100 mg liter and displayed reversibility similar
to that of Sortin1 (Fig. 2E).
Sortin1 Stimulates Protein Secretion in Arabidopsis Suspension Cells.
To examine the effect of Sortin1 and Sortin2 on secretion at the
biochemical level, we treated plant suspension cells with the
drugs. Cell viability was first examined at several concentrations
over time in Arabidopsis and tobacco BY2 cells by using the vital
dye fluorescein-diacetate (data not shown). When compared
with untreated control cells (Fig. 3 A and D), Sortin1 was well
tolerated at a concentration of 25 mg liter (57 M) for 16 h
by both Arabidopsis and tobacco cells (Fig. 3 B and E). To the
contrary, Sortin2 was highly toxic, and the cells were found
without metabolic activity at a concentration of 25 mg liter (58
M) after 8 h (Fig. 3 C and F). Cell death was also apparent at
doses of Sortin2 as low as 10 mg liter (23 M) after only 4 h (data
not shown). The similar results from Arabidopsis and tobacco
cultured cells indicated that the toxic effect of the drugs was not
specific to cells from a single plant species. Due to the high
toxicity of Sortin2 on cultured cells, and thus the possibility of
cell lysis, we focused on Sortin1 to ask whether a sublethal dose
would result in a significant increase in overall protein secretion.
Fig. 2. Sortin1 and Sortin2 alter vacuolar morphology in Arabidopsis seedlings.
Seedlings expressing EGFP: -TIP as a tonoplast marker were germinated
in the absence of Sortins (A) or in the presence of 227 M Sortin1 (B) or 233 M
Sortin2 (C). Vacuolar morphology in hypocotyls of 1-week-old seedlings was
examined by confocal microscopy. Plants were then transferred onto nondrug
medium to demonstrate reversibility of the phenotype induced by
Sortin1 (D) and Sortin2 (E). Images in D and E (left to right) were taken at 0, 1,
2, 3, 4, and 5 days after transfer to non-drug medium. (Scale bars 20 m in
A­C and 3 mm in D and E.)
Fig. 3. Sortin1 induces secretion of the AtCPY precursor in Arabidopsis cell
suspensions. The viability of Arabidopsis (A­C) and tobacco BY2 (D­F) suspension
cells that were untreated (A and D) or treated with 57 M Sortin1 (B and
E) or 58 M Sortin2 (C and F) was analyzed by using fluorescein-diacetate. (G)
Proteins secreted into the growth media of Arabidopsis suspension cells that
were untreated (lane 1) or treated with Sortin1 (lane 2) were concentrated
and analyzed by SDS PAGE. Growth medium samples were also analyzed by
Western blot (control, lane 5; Sortin1, lane 6). The AtCPY processing pattern
was examined in cell pellets (control, lane 3; Sortin1, lane 4). The positions of
the 60-kDa precursor (p), 48-kDa intermediate (i), and 24-kDa mature (m)
forms of AtCPY are indicated. (H) A pulse­chase experiment tracking the
AtCPY precursor secretion in cells that were untreated (control) or treated
during the chase period with Sortin1 (Sortin1). (Scale bar 500 m in A­F.)
Zouhar et al. PNAS June 22, 2004 vol. 101 no. 25 9499
PLANTBIOLOGY
After treatment with 57 M Sortin1 for 16 h, we concentrated
the growth media and separated the proteins by SDS PAGE.
The protein profiles in Coomassie blue-stained gel lanes from
untreated and treated Arabidopsis cells (Fig. 3G, lanes 1 and 2,
respectively) differed significantly, suggesting that Sortin1 triggered
a general increase in protein secretion. AtCPY is known
to undergo processing after delivery to the vacuole (22), so we
assayed for intracellular and extracellular mature and precursor
forms of AtCPY to provide direct evidence of altered fidelity of
protein sorting. Indeed, when we analyzed the concentrated
growth media by immunoblots with anti-CPY antibodies, we
identified a precursor of AtCPY in medium from cells treated
with Sortin1 (Fig. 3G, lane 6) that was absent in an untreated
control sample (Fig. 3G, lane 5). Our data indicated that a
notable amount of AtCPY precursor was diverted to the secretion
pathway as a result of Sortin1 treatment. The lack of mature
AtCPY in the medium indicated that AtCPY detected in the
medium was probably the result of active secretion and not cell
and vacuole lysis. A similar analysis of cell pellets did not reveal
any obvious difference in intracellular processing of AtCPY (Fig.
3G, lanes 3 and 4). This finding indicated that, at the sublethal
dose examined, not all AtCPY was secreted. However, the
proportion that was secreted was diverted at a step before
proteolytic processing. This finding also pointed to the fine level
of control that was possible with Sortin1 derived from the
chemical screen.
To demonstrate conclusively that Sortin1 induced secretion of
the AtCPY precursor in metabolically active cells, we performed
a pulse­chase experiment in Arabidopsis suspension cells in the
presence of Sortin1. Cells were labeled with 35
S-amino acids and
chased with unlabeled amino acids in the presence or absence of
Sortin1. The AtCPY that was secreted into the media was then
immunoprecipitated by using anti-CPY antibodies and analyzed
by SDS PAGE and fluorography. Cells treated with Sortin1
displayed a rapid accumulation of a polypeptide corresponding
to the unprocessed precursor of AtCPY. Compared with an
untreated control in which little or no precursor was detected
even after 24 h (Fig. 3H, control), this accumulation was
detectable in 3 h or less after Sortin1 treatment (Fig. 3H,
Sortin1). These results clearly established that Sortin1 stimulated
secretion from cells that were metabolically active and intact.
Sortin1 Stimulates Secretion in Whole Plants. To investigate the
effect of Sortin1 in intact plants, we performed immunoelectron
microscopy of AtCPY in 1-week-old Arabidopsis seedlings grown
in the presence of the drug at a concentration of 57 M.
Compared with untreated controls (Fig. 4 A and G), immunogold
labeling detected a significant amount of AtCPY in the
apoplast of hypocotyl (Fig. 4B) and root (Fig. 4H) tissue of
treated seedlings. Consistent with the results of the immunoblots,
AtCPY was also detected in vacuoles of untreated and
treated seedlings (Fig. 4 D and E, respectively), again indicating
that, at sublethal doses, redirection to the secretion pathway was
not complete. We also performed immunolocalization of the
vacuolar invertase AtFruct4 (22) in root tissue of Sortin1-treated
Arabidopsis seedlings. In young seedlings, AtFruct4 was previously
localized to endoplasmic reticulum (ER)-derived precursor
protease vesicles (22) and thus utilizes a different pathway
than AtCPY for vacuolar targeting. Labeling of AtFruct4 was
clearly observed in precursor protease vesicles of 1-week-old
seedlings treated with Sortin1 (Fig. 4I). Interestingly, no AtFruct4
labeling in the apoplast was detected (Fig. 4I), suggesting
that Sortin1 probably targets specific protein sorting pathways.
In addition, when we analyzed the concentrated growth medium
from cultured Arabidopsis cells treated with Sortin1, no vacuolar
invertase was detected, which is consistent with our immunolocalization
and cell viability assays (data not shown). Several
drugs known to affect endomembrane trafficking have significant
impacts on Golgi morphology (reviewed in ref. 29). To
further characterize the effect of 57 M Sortin1 on the endomembrane
system, we examined the morphology of the Golgi
apparatus (Fig. 4J Left) and ER (Fig. 4J Right) in hypocotyls of
1-week-old seedlings by electron miscroscopy. Sortin1 treatment
did not affect Golgi structure or trigger loss of cisternae.
Additionally, 57 M Sortin1 did not alter the morphology of
either the ER or the central vacuole in seedlings (Fig. 4K) or
Arabidopsis suspension cells (Fig. 4L).
Discussion
We have taken advantage of similarities in the secretion pathways
in yeast and plants to screen for chemicals that stimulate
secretion. Of the 14 confirmed compounds from our initial yeast
screen, two were found to be active in plants and to affect vacuole
biogenesis and root development in Arabidopsis seedlings. The
severe yet reversible effects of Sortin1 and -2 on root development
may be due to the essential nature of vacuole biogenesis in
plants. For example, the vcl1 mutant of Arabidopsis lacks proper
vacuole development, missorts vacuole proteins to the extracelFig.
4. AtCPY is secreted into the apoplast in Sortin1-treated 1-week-old
Arabidopsis seedlings. Immunolocalization of AtCPY was performed in sections
of control plants (hypocotyl, A and D; root, G) or Sortin1-treated plants
(hypocotyl, B and E; root, H). A preimmune serum was used as a control (C and
F). Immunolocalization of invertase AtFruct4 was performed in Sortin1treated
roots (I). A­C and G­I show apoplast whereas D­F are of vacuoles.
Arrowheads indicate the position of gold particles. Golgi (g) (J Left) and ER
(J Right) morphology was examined in 1-week-old Sortin1-treated seedlings
by electron microscopy. Tonoplast morphology in 1-week-old seedlings expressing
EGFP: -TIP and treated with Sortin1 was analyzed by confocal microscopy
(K). Arabidopsis suspension cells were treated with Sortin1 for 16 h
and examined by brightfield light microscopy (L). (Scale bars 200 nm in A­J
and 20 m in K and L.)
9500 www.pnas.org cgi doi 10.1073 pnas.0402121101 Zouhar et al.
lular space, and is embryo lethal (3). Thus, it is reasonable that
drugs that disrupt these processes would lead to growth defects.
The severity of root phenotype was likely enhanced by direct
contact of the developing roots with the drug-containing growth
media.
One of the compounds, Sortin1, was examined in detail and
found to stimulate protein secretion in general and AtCPY in
particular in both cell cultures and the whole plants. We have
demonstrated the validity of using a simple eukaryote to screen
for drugs that affect the plant endomembrane machinery, which
reflects evolutionary similarities. However, the fact that not all
of the chemicals were biologically active points to significant
differences between yeast and multicellular plants. Such differences
could be in uptake, intercellular transport, or metabolism
of the drugs due to the complex multicellular nature of plants
compared with yeast and other evolutionary differences in the
sorting machineries. Thus, the use of yeast must be viewed as a
complement to plant-based screens.
The few drugs that have been available that affect the endomembrane
system including the Golgi-disturbing drugs brefeldin
A and monensin (reviewed in ref. 29), the N-glycosylation
inhibitor tunicamycin (reviewed in ref. 30), and the recently
identified Exo1 (27) have been extremely useful in increasing our
understanding of the endomembrane system even though all of
their cognate targets are still unclear (27, 31). Recognizing the
power of such reagents, we have used a chemical genomics
approach to identify several sorting drugs termed Sortins. In
contrast to brefeldin A and Exo1, which interfere with exocytosis,
Sortins seem to stimulate secretion of AtCPY and other
proteins but not proteins delivered to the vacuole by means of
protease precursor vesicles. The ionophore monensin has been
shown to stimulate protein secretion in cotelydons (32, 33).
However, it is unlikely that a mode of action of Sortin1 is
analogous to that of monensin. At the cellular level, the most
dramatic phenotypes resulting from monensin treatment are
swollen Golgi cisternae. We examined the Golgi structure by
electron microscopy after Sortin1 treatment and found that the
cisternae stack was intact. This result indicates that Sortin1
probably does not act as a monensin-like ionophore and functions
through a different mode of action. We examined the
activity of Sortin1 at a sublethal dose. Given that vacuole
biogenesis is essential in plants (3), the ability to ``fine tune'' the
biological response is a critical advantage over the use of
conventional genetics in which such adept control is seldom
possible.
Although we do not yet know the cognate target of Sortin1, the
general stimulation of protein secretion and the secretion of
AtCPY precursor, as well as aberrant vacuole biogenesis, suggest
that the drug acts at a step before proteolytic processing and may
interfere with vesicle budding or fusion or other essential process
in vacuole biogenesis. One of the major challenges of the
chemical genomics approach is target identification. Arabidopsis
is very well suited for identification of putative targets by
isolation of either resistant or hypersensitive mutants to a
particular compound (10). Arabidopsis has also proven to be a
useful organism for the identification of a potential target for
a specific bioactive compound (6). A powerful advantage of
chemical genomics not often recognized is the ability to challenge
characterized mutants with drugs like Sortin1 to look for
interactions within a pathway or with interacting pathways. In
addition, the use of chemical analogues with altered or no
biological activity could provide correlations between structure
and activity. Such analyses will facilitate target identification and
provide additional insights into the mechanics of endomembrane
transport.
We thank Dr. Valentina Kovaleva for skilled immunolocalization analyses.
We thank members of the Raikhel laboratory for helpful discussions.
Special thanks are due to Dr. Thomas Girke for handling the
compound library and Ms. Jocelyn Brimo for formatting the manuscript.
This work was supported by National Science Foundation Grant MCB0296080
(to N.V.R.).
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