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First published online November 7, 2006
doi: 10.1242/10.1242/dev.02654
1 University of Köln, Botanical Institute III, Gyrhofstr. 15, 50931
Köln, Germany.
2 MEDICAGO Inc., 1020, route de l'Église, bureau 600, Sainte-Foy,
Québec G1V 3V9, Canada.
3 Laboratoire de Génétique Moléculaire des Plantes,
CNRS/Universite J. Fourier BP 53, 38041 Grenoble Cedex 09, France.
4 Centre for Molecular Medicine Cologne, University of Cologne,
Joseph-Stelzmann-Str. 52, 50931 Köln, Germany.
5 Institute of Biochemistry II, Medical Faculty, University of Cologne,
Joseph-Stelzmann-Str. 52, 50931 Köln, Germany.
* Authors for correspondence (e-mail: swen.schellmann{at}uni-koeln.de; martin.huelskamp{at}uni-koeln.de)
Accepted 20 September 2006
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS The
DISCUSSION
REFERENCES |
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Key words: Endosome, ESCRT, Mono-ubiquitylation, Multivesicular body, Protein degradation, Arabidopsis
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS The
DISCUSSION
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;
Vierstra and Callis, 1999).
Proteins that are subject to degradation are linked to chains of several
ubiquitin molecules by a cascade of E1, E2 and E3 enzymes.
Poly-ubiquitin-marked proteins are targeted to the proteasome, a large complex
where the protein is degraded.
Recently, an alternative ubiquitin-dependent route for protein degradation
was discovered (Babst, 2005;
Katzmann et al., 2001). In
this pathway, proteins labeled by a single ubiquitin are targeted to the
lysosome (animals) or vacuole (yeast)
(Babst, 2005). This pathway is
also used for the delivery of proteins (e.g. proteases) that normally reside
in the vacuole (Babst et al.,
2000).
The initial step is the recognition of mono-ubiquitlyated proteins by
Vps23p and Vps27p (Bilodeau et al.,
2002; Bilodeau et al.,
2003). Vps23p is a component of the ESCRT I complex that is
located at the late endosome, the multivesicular body (MVB). The MVB contains
numerous vesicles that originate from the invagination of the outer membrane.
Proteins targeted to the MVB membrane by Vps23p and Vps27p become internalized
with the aid of the ESCRT II and ESCRT III complexes
(Babst et al., 2002a;
Babst et al., 2002b;
Katzmann et al., 2001). The
MVBs eventually fuse with the vacuole/lysosome where proteins are degraded by
luminal proteases (Odorizzi et al.,
1998).
This protein degradation pathway appears to specifically recognize proteins
linked to a single ubiquitin. This is evident from the finding that pCPS, a
precursor of carboxypeptidase S, is misguided to the vacuolar membrane when
the N-terminal lysine to which ubiquitin is bound is replaced by other amino
acids (Katzmann et al., 2001).
Conversely, proteins that are not normally targeted to the vacuole were
transported into the vacuole when linked to single ubiquitin molecules
(Urbanowski and Piper, 2001).
This pathway is also found in mammals. Homologs of all the components of yeast
ESCRT complexes have been found, and the human tumor susceptibility gene 101
protein (TSG101) was shown to be the ortholog of Vps23p
(Babst, 2005). Individuals
deficient in TSG101 develop breast cancer, and mutant cell lines show mitotic
defects including aberrant mitotic spindles, multiple nuclei and nuclear
anomalies (Xie et al.,
1998).
The yeast and plant vacuole and the animal lysosome can be considered
functionally similar compartments. All are characterized by an acidic pH and
the existence of proteases. Vacuolar protein degradation in plants has been
studied in recent years (Vitale and
Galili, 2001; Vitale and
Raikhel, 1999), but was not linked to the mono-ubiquitin-mediated
pathway. In this study, we describe the isolation and functional
characterization of the ELCH (ELC) gene. The elc
mutant was initially identified because of a trichome morphogenesis phenotype.
A closer analysis showed that elc mutants have multiple nuclei, not
only in trichomes but also in all endoreduplicating cell types analyzed. The
ELC gene encodes a protein with sequence similarity to yeast Vps23p
and human TSG101, which are components of the ESCRT I-III protein sorting
machinery. All known components of the ESCRT I-III complexes are found in the
Arabidopsis genome, indicating that the mono-ubiquitin-dependent
vacuolar protein degradation pathway is conserved between plants, yeast and
animals. The ELC protein binds ubiquitin in vitro. Gel filtration assays
suggest that ELC is part of a large protein complex, and yeast two-hybrid and
co-immunoprecipitation assays show that ELC binds to Arabidopsis
VPS37 and VPS28. A YFP:ELC fusion protein is localized to endosomal
compartments. We show that tubulin folding cofactor a mutants
synergistically enhance the phenotype, suggesting that ELC function is linked
to microtubules.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
RESULTS The
DISCUSSION
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Constructs and recombinant DNA
The rescue of the elc mutant was performed with a fragment
amplified from the P1 clone MQC3 using Expand High Fidelity Polymerase (Roche)
and the following primers: CS023,
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAAGCAGGAGTGTCTAGG-3'; and CS038,
5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGTTGAGGAATGTATGGGC-3'. The PCR
product was gel purified and cloned into the Ecl136II restriction
site of pCAMBIA-1300 (GenBank accession number AF234296). The 35S::ELC-HA
fusion was generated using the following primers: CS063,
5'-AAGGGCCCGTCGACCATGGTTCCCCCGCC-3'; and CS064,
5'-TAGGGCCCGCGGCCGCTCAAGCGTAATCTGGAACATCATATGGGTACCTACCTGCGATGGCTGC-3',
which includes the sequence of the HA-tag. The PCR product was subcloned into
pGEMT®-easy (Promega), excised with SalI and NotI,
blunt-ended and then ligated into the SalI site of pBinAR
(Hofgen and Willmitzer,
1990).
The cDNA for Arabidopsis VPS37-1 was obtained from RIKEN Genomic
Sciences Centre (Seki et al.,
1998; Seki et al.,
2002).
Flanking genomic sequences of the T-DNA insertions were isolated using Vectorette PCR. The position of the T-DNA insertion was studied by TAIL-PCR using nested primers annealing within the coding sequence:
AS-TAIL-1, 5'-TTTATGCACAACGATGGTCGCTCCG-3';
AS-TAIL-2, 5'-CGCACATGTCACTCCTTCTGGTCTCGTT-3'; and
AS-TAIL-3, 5'-AGACCGTTCCCGCCATCACCTTACG-3'.
For the RT-PCR analysis, primers were chosen from regions where the ELC gene and its close homolog At5g13860 show significant differences:
AS-ELCHRT-fw1, 5'-ACACCGTTTAACTCCGATGGTTCCCCC-3';
AS-ELCHRT-rev1, 5'-GGTGGTGAACATGCTGCACTTGCACCC-3';
AS-ELCHHOMRT-fw, 5'-GTCATCTGTCGTCGTCTTCAAGTAATCG-3'; and
AS-ELCHHOMRT-Rrev, 5'-TGTATATTCCTATACCCAATAAAAATCG-3'.
As neither gene contains introns, an RT reaction without reverse transcriptase was performed as a control for genomic contamination. To calibrate cDNA amounts, actin expression was determined using primers: AS-Act-RT3, 5'-GGATAGCATGTGGAAGTGCATAC-3'; and AS-Act-RT5, 5'-TGCGACAATGGAACTGGAATG-3'.
Ubiquitin binding assay
Crude protein extracts were made from 14-day-old transgenic plants
expressing the 35S:ELC-HA construct using a protein extraction buffer (50 mM
phosphate, 150 mM NaCl, 5 mM DTT) and Complete Protease Inhibitor (Roche)
according to the manufacturer's instructions. Crude extracts were purified by
centrifugation at 16 000 g at 4°C for 5 minutes.
Ubiquitin-agarose beads (Sigma) and Protein G-agarose beads (Roche) were equilibrated in extraction buffer and an equal volume of plant extract was added. After 2 hours at 4°C with slight agitation, the beads were centrifuged at 400 g and washed two to three times with extraction buffer. Beads and washing fractions were incubated with loading buffer at 99°C for 10 minutes. The proteins were separated by SDS-PAGE and detected by western blotting. The HA epitope was detected with rat anti-HA monoclonal antibody (Roche). An HRP-linked goat anti-rat secondary antibody was used (Jackson Immuno Research Laboratories).
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).
Immunoprecipitation of ESCRT I complex
Protein extracts were made from plants overexpressing the ELC-HA construct
under control of the CMV 35S promoter. Plants were grown on hygromycin (25
µg/ml) for 14 days at 22°C under long-day conditions. Plants (0.9 g)
were ground in 1.4 ml lysis buffer [50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1%
Triton X-100] complemented with 100 µl protease inhibitor (one tablet
Complete Protease Inhibitor in 2 ml lysis buffer) and 17.5 µl 1 M DTT. A 1
ml aliquot of this lysate was used with 120 µl anti-HA MicroBeads (Miltenyi
Biotec) according to the manufacturer's instructions with the following
modifications: the column was rinsed five times with 200 µl Wash Buffer 2,
and the protein was eluted with 60 µl Elution Buffer. PAGE was performed
using 12% polyacrylamide gels.
Protein identification by peptide mass fingerprinting
After PAGE, the gel was silver-stained as described previously
(Blum et al., 1987). Bands
were excised and analyzed by MALDI-TOF mass spectrometry (Bioanalytics Centre
for Molecular Medicine, Cologne, CMMC).
Protein bands were excised from the gel, treated three times with acetonitrile:water (1:1), once with acetonitrile, reswollen in 50 mM NH4HCO3 and dried in a speedvac. Then, 10 mM DTT in 50 mM NH4HCO3 was added and the proteins reduced for 45 minutes at 56°C. To alkylate reduced cysteine residues, the remaining liquid was removed and the probe incubated with 50 mM iodoacetamide in 50 mM NH4HCO3 for 30 minutes in the dark. Thereafter, the gel pieces were washed and dried as above. The gel pieces were treated for 1 hour with 12.5 ng/µl trypsin (sequencing grade, Promega) in 25 mM NH4HCO3, 10% acetonitrile, then washed in the same buffer without enzyme and the proteins digested at 37°C overnight. The digest was stopped by the addition of 5 to 20 µl 1% trifluoroacetic acid (TFA), and the peptides extracted for 30 minutes at 37°C.
A 1.0 µl aliquot of the extracted peptides was mixed with 1.2 µ l 2.5 mg/ml 2,5-dihydroxybenzoic acid in 0.1% TFA:acetonitrile (2:1) and spotted onto a 800 µm anchor target (Bruker Daltonics, Bremen, Germany). Positive ion spectra were acquired on a Reflex IV MALDI-TOF mass spectrometer (Bruker Daltonics) in the reflectron mode. A peptide calibration standard (Bruker Daltonics) was used for external calibration of the mass range from m/z 1046 to m/z 3147. Proteins were identified from MALDI fingerprint data by searching the NCBInr public database (release 20060315) using a local installation of MASCOT 1.9.
Cell culture, protoplast isolation and transfection
Arabidopsis cell suspension culture (Columbia ecotype; grown in MS
medium supplemented with 0.5 mg/l NAA and 0.1 mg/l KIN) was maintained as
described (Mathur and Koncz,
1998). Protoplast isolation and polyethylene glycol-mediated
transfection were performed according to Magyar and co-workers
(Magyar et al., 2005). The
transfected cells were incubated at 23°C for 16 hours in the dark before
staining and microscopic observation.
For YFP and FM4-64 double-labeling and co-transfection, YFP-ELC was
introduced into the protoplasts of Arabidopsis suspension culture
cells as described above. After 16 hours incubation in MS cell culture medium
containing 0.34 M glucose, 0.34 M mannitol, protoplasts were collected by
centrifugation, resuspended in the same medium containing 50 µM FM4-64, and
placed on ice for 10 minutes (Ueda et al.,
2001). After labeling, cells were washed twice, resuspended in the
same medium without FM4-64 and analyzed.
Microscopy and cell biology
The C value of trichome nuclei was determined using the DISCUS software
package (Carl H. Hilgers-Technisches Büro, Königswinter, Germany).
The fluorescence intensity of DAPI-stained nuclei was determined, and the
relative fluorescence units (RFU) were calibrated with stomata trichome nuclei
that have a DNA content of 2C (Galbraith
et al., 1991). Light microscopy was performed with a Leica DMRE
microscope (Leica, Wetzlar, Germany) equipped with a high-resolution
KY-F70-3CCD JVC camera and frame-grabbing DISKUS software. Confocal microscopy
was performed using a TCS SP2 (Leica). Leaf sections were stained with 500
µg/ml propidium iodide and infiltrated for 15 minutes at 0.9 bar. After
staining, leaves were embedded in 5% low-melting-point agarose and sectioned
with a razor blade. Images were processed using Adobe Photoshop 6.0
software.
Yeast transformation and yeast two-hybrid assay
The yeast strain AH109 (Halladay and
Craig, 1996) was grown in standard yeast full media or selective
drop-out media (Clontech) under standard conditions.
Transformation of plasmids into yeast was performed using the Lithium
acetate method (Gietz et al.,
1995). Interactions were analyzed on synthetic drop-out medium
lacking leucine and tryptophan and on synthetic drop-out medium without
leucine, tryptophan and histidine supplemented with 3 mM 3-aminotriazole
(3-AT) (Sigma-Aldrich, Munich, Germany) after 3 days of growth. SNF1 and SNF4
were used as positive controls (Fields and
Song, 1989). The interaction between ELC and VPS37-1 was observed
in eight independent experiments.
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SUMMARY
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MATERIALS AND METHODS
RESULTS The
DISCUSSION
REFERENCES |
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). When tracing the path of single peroxisomes we found them
moving from one stem into the other, demonstrating that the two stems belong
to one cell (Fig. 1E-H).
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). If
ELC acts at the switch from mitosis to endoreduplication, either of
two scenarios could occur in elc mutants. First, an incomplete cell
division could take place instead of the first endoreduplication cycle. In
this case, the total DNA content of each of the two nuclei should be 16C and
the total cellular DNA content should be 32C. Second, the first incomplete
cell division might be followed by the normal trichome differentiation
program; in this case, each of the two nuclei would be 32C and the total DNA
content would be doubled. When measuring the DNA content of nuclei in
elc mutants, we found that individual nuclei in elc
trichomes containing one or two nuclei had a DNA content of approximately 32C
(Fig. 3), suggesting that the
second scenario is true.
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The ELC gene has no introns and shows sequence similarity to the
yeast Vps23 and mammalian TSG101 genes (60% positives and 11%
identical, amino acid level) (Fig.
4D). Although the overall similarity is not high, the three genes
do share a similar size and arrangement of domains
(Fig. 4E). At the N-terminus,
all three share a ubiquitin conjugating enzyme variant (UEV) domain that is
missing a cysteine conserved in ubiquitin conjugating (UBC) domains. According
to the COILS program (Lupas et al.,
1991), a coiled-coil region is located in the middle part of the
protein. At the C-terminus is located a conserved domain termed the steadiness
box because it is involved in the control of the stability of TSG101
(Feng et al., 2000). The
Arabidopsis genome contains, in addition to ELC, a close
homolog (72% identity) with the same domain structure and without the crucial
cysteine in the UEV domain. ELC homologs are also present in
monocotyledons such as rice (Fig.
4D).
The expression of ELC was determined by RT-PCR. Expression was found in all tissues analyzed including roots, stems, leaves and flowers (Fig. 5A). These results are consistent with the expression data published in GENEVESTIGATOR (www.genevestigator.ethz.ch).
The genes of the ESCRT I-III pathway are conserved in plants
In yeast, Vps23p is part of a mono-ubiquitylation-dependent protein sorting
pathway in which Vps23p binds ubiquitin through its UEV domain, which shows
sequence similarity to E2 conjugating enzymes
(Katzmann et al., 2001).
Vps23p is part of the ESCRT I complex which consists of three subunits,
Vps23p, Vps28p and Vps37p, which belong to the class E vacuolar sorting
proteins (Bankaitis et al.,
1986; Banta et al.,
1988; Odorizzi et al.,
1998; Raymond et al.,
1992). Subsequently, further downstream class E Vps proteins are
activated that form the ESCRT II and ESCRT III complex that mediate the actual
protein sorting (Babst et al.,
2002a; Babst et al.,
2002b; Katzmann et al.,
2001). A detailed database search revealed that homologs of all
components of the ESCRT I, II and III complexes are present in the
Arabidopsis genome (Table
3).
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ELC is a component of a high-molecular-weight protein complex
The yeast homolog of ELC, Vps23p, has been found in a large complex of
about 350 kDa (Babst et al.,
2000). In order to test whether the plant homolog is also part of
a complex, we used the ELC-HA transgenic lines described above. Upon
separation on a Superose 6 10/300 GL gel filtration column, we detected ELC-HA
in a size range between 200 and 400 kDa
(Fig. 7).
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These data are consistent with studies in mammals where VPS37 is known to
interact with TSG101 (Bache et al.,
2004; Eastman et al.,
2005; Stuchell et al.,
2004). The interaction between VPS37-1 and ELC was additionally
confirmed by a yeast two-hybrid assay using both genes as bait and as prey. As
summarized in Table 4,
interactions were found between the two proteins. In summary, these data show
that ELC is part of the plant ESCRT I complex.
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). As shown in Fig.
9, the small compartments showing YFP-ELC staining were also
stained with FM4-64. This demonstrates that ELC is localized to the endosomes.
We also found FM4-64-labeled compartments without YFP fluorescence, indicating
that ELC is not present in all endosomal compartments
(Fig. 9A-D). To determine more
specifically the stages at which ELC is localized to the endosome, we
co-transfected CFP-ELC with Ara6-GFP, as a marker for early endosomes, or with
GFP-Ara7, as a marker for late endosomes
(Ueda et al., 2001). We found
co-localization of CFP-ELC with both Ara6-GFP
(Fig. 9E-H) and GFP-Ara7
(Fig. 9I-L), indicating that
ELC is localized to early and late endosomes.
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;
El Refy et al., 2004). The
kak elc double mutant exhibited an additive trichome phenotype. The
cluster frequency and number of nuclei was similar to that in elc
mutants (Table 1) and the
number of branches was similar to that in kak mutants. Thus,
additional endoreduplication cycles do not lead to more nuclei in the
elc mutant background, suggesting that the ELC gene is not
important during endoreduplication cycles but at the initial switch from
mitosis to endoreduplication. The additive phenotype provides no hint towards
a redundant function of the ELC-dependent pathway and the putative E3 ligase
KAKTUS-dependent proteasome pathway.
Genetic evidence suggests that ELC controls nuclear divisions through the microtubule cytoskeleton
Several pathways are known by which the ESCRT I-III pathway controls the
cell cycle in animals. However, the absence of the relevant components of
these pathways renders it unlikely that these pathways are operating in plants
(for details see Discussion). Because several examples are known in which
microtubule misregulation results in multinuclear cells in plants
(Kirik et al., 2002a;
Kirik et al., 2002b;
Muller et al., 2004;
Steinborn et al., 2002), we
speculated that ELC controls nuclear divisions through the microtubule
cytoskeleton. To test this hypothesis, we created an elc kiesel-T1
(kis-T1) double mutant. The KIS gene encodes a homolog of
tubulin-folding cofactor A, a component of a pathway important for the
formation of assembly competent 
/ß tubulin dimers
(Kirik et al., 2002a;
Steinborn et al., 2002).
Strong KIS mutants have multiple nuclei and are embryo lethal
(Steinborn et al., 2002). We
used the weak kis-T1 mutant that displays a mild growth
defect, slightly reduced cell division frequency, reduced trichome branching
and occasionally cells with multiple nuclei
(Kirik et al., 2002a). The
double mutant showed a synergistic phenotype: the frequency of trichomes with
more stems was drastically increased (16%; n=743) as compared with
elc (2%; n=3529) and kis-T1 (0.8%; n=1356)
(Fig. 10A-F). Epidermal
pavement cells were strongly reduced in size
(Fig. 10G-I), the leaf
architecture was grossly disturbed (Fig.
10J-L) and many cells contained multiple nuclei and nuclear
anomalies (Fig. 10M-O). The
strong mutual enhancement of the phenotypes indicates that the two genes act
in the same pathway and hence that ELC regulates cell division at least in
part through the microtubule cytoskeleton.
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DISCUSSION
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;
Urbanowski and Piper, 2001).
In this pathway, mono-ubiquitylated proteins are targeted to the late
endosome, the so-called MVB. After fusion of the MVB with the vacuole (in
yeast) or the lysosome (in animals), proteins become accessible to proteases
and are degraded. A regulation of the cell cycle by this pathway is suggested
by the finding that mutations in human TSG101, which is involved in sorting
mono-ubiquitylated proteins into the MVB
(Katzmann et al., 2001), lead
to mitotic defects including multiple MTOCs (microtubule organizing centres),
misdistribution of chromosomes and multiple nuclei
(Xie et al., 1998). Our
finding that mutations in the Arabidopsis homolog of TSG101 lead to
multiple nuclei suggest that a related pathway is operating in plants.
Non-proteasomal protein degradation in lytic vacuoles in plants
Although the Vps23p/TSG101 pathway has not been described in plants, it has
been shown that lytic vacuoles similar to those in yeast exist
(Swanson et al., 1998). Plant
cells can have two different types of vacuoles
(Paris et al., 1996), a lytic
and a protein storage vacuole (Swanson et
al., 1998; Vitale and Raikhel,
1999). These two different vacuoles seem to fuse in mature cells
to form the central vacuole. Newly synthesized proteins enter different routes
for the lytic and the storage vacuoles
(Bassham and Raikhel, 2000).
Proteins destined for the storage vacuole are found in PACs (precursor
accumulating vesicles) before they reach the vacuole. Alternatively, proteins
may be routed via DVs (dense vesicles) and the MVB
(Tse et al., 2004). Proteins
targeted to the lytic vacuole are found in the CCV (clathrin-coated vesicles)
and the PVC (prevacuolar compartment) before they reach the lytic vacuole.
The ESCRT-pathway in plants
To date, there is no biochemical evidence for the existence of a
mono-ubiquitin-dependent protein-sorting pathway in plants. At present, this
excludes the possibility to directly test a function of ELC in protein
sorting. However, the overall sequence similarity and the conserved
arrangement of domains suggest that ELC encodes a Vps23p/TSG101
homolog. That ELC acts in a Vps23p/TSG101 pathway is indicated by
four observations. First, the complete ESCRT machinery known from yeast is
conserved in Arabidopsis. All three subunits of the ESCRT I complex,
Vps23p, Vps28p and Vps37p, were found. The three subunits comprising the ESCRT
II complex, Vps22p, Vps25p and Vps36p, and those of the ESCRT III complex,
Vps2p, Vps20p, Vps24p and Snf7, are all present in the Arabidopsis
genome (see also Winter and Hauser,
2006). Second, we found that the ELC protein binds ubiquitin in
vitro. Third, ELC is part of a high molecular weight complex and binds
directly to VPS37 and VPS28, two components of the ESCRT I complex, as has
been shown for the mammalian TSG101 (Bache
et al., 2004; Eastman et al.,
2005; Stuchell et al.,
2004) and for Vps23p in yeast
(Katzmann et al., 2001).
Forth, YFP:ELC co-localizes with the endocytotic markers FM4-64
(Vida and Emr, 1995), ARA6 and
ARA7 (Ueda et al., 2001),
indicating that ELC localizes to early and late endosomes.
|
). The
molecular link to the cell cycle is still unclear and three possibilities that
are not mutually exclusive are currently discussed: (1) Because it has been
shown that cell-cycle relevant receptors (e.g. the EGF-receptor) are subject
to TSG101-dependent degradation, it is assumed that the cell division
phenotype is caused by a failure to degrade these receptors
(Babst et al., 2000;
Carter and Sorkin, 1998); (2)
A second possibility is that TSG101 is involved in the control of the known
cell cycle regulator p53. It has been shown that TSG101 is part of a
regulatory circuitry with MDM2 and p53
(Haupt et al., 1997;
Kubbutat et al., 1997;
Li et al., 2001); (3) A third
possibility is that TSG101 controls the stability of p21Cip1
(CDKN1A - Human Gene Nomenclature Database), which is a well-characterized
inhibitor of CDK activity. It has been shown that TSG101 interacts with
p21Cip1 in the yeast two-hybrid assay
(Oh et al., 2002) and that the
stability of p21Cip1 was drastically increased in the presence of
TSG101.
It is unlikely that these pathways are operating in plants. A true p53 or
EGF ortholog has not yet been found in the fully sequenced
Arabidopsis genome. Also, a clear p21Cip1 ortholog is
missing. Instead, a class of ICK/KRP proteins sharing a short motif
responsible for CDK and cyclin binding exists
(Verkest et al., 2005). It is
therefore likely that cell division regulation by Vps23p/ELC/TSG101 is
mediated by an as yet undescribed route in this context. Our genetic data
provide a first hint. The elc phenotype is reminiscent of mutants
defective in regulators of the microtubule cytoskeleton
(Kirik et al., 2002a;
Kirik et al., 2002b;
Muller et al., 2002;
Muller et al., 2004;
Steinborn et al., 2002;
Twell et al., 2002) and is
enhanced in the elc kis-T1 double mutant. This suggests that the
regulation of the microtubule cytoskeleton by ELC might be relevant
for the regulation of cell division in plants.
Perspectives
Although the phenotype of the first plant mutant in the ESCRT I-III pathway
indicates that this pathway is important for the regulation of cytokinesis, it
is likely that this occurs through different routes than in animals. It will
be imperative to study the ESCRT I-III pathway in plants in more detail.
Markers are required to trace the fate of proteins in this pathway. The key
challenge will be the identification of proteins that are regulated by this
pathway.
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ACKNOWLEDGMENTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS The
DISCUSSION
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