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INTRODUCTION |
RNA localization and spatial restriction of translation are
important mechanisms in biological processes (reviewed in Refs. 1 and
2). The process of mRNA localization is initiated by association of
mRNA with one or more RNA-binding proteins through a targeting
signal most commonly located in the 3'-untranslated region of the
transcripts. This association may occur in the nucleus soon after
transcription, resulting in the formation of ribonucleoprotein particles (3), which are then exported into the cytosol (4). Alternatively, such complexes may form in the cytosol. RNP particles migrate along the cytoskeleton to their final destinations, where they
are anchored and translated (reviewed in Ref. 5). The multistep process
of RNA localization depends on specific trans-acting proteins. One
common feature of these factors is repeats of different RNA binding
domains (6). These RNA-binding proteins include members of the
double-stranded RNA-binding protein family (7, 8), homologues of the
zipcode-binding protein (9, 10), and members of the heterogenous
nuclear RNP1 family
(11-13).
One of the most common protein motifs involved in RNA binding is the
K-homology (KH) domain, originally described in the protein heterogenous nuclear RNP-K (14). A KH domain consists of ~70 amino
acids and includes a conserved hydrophobic core, an invariant GXXG motif, and an additional variable segment. NMR
structural studies of individual KH domains revealed a conserved





fold (15, 16).
In yeast, Scp160p, a protein containing 14 copies of the KH domain, has
been identified (17). Scp160p localizes to the cytosol with an
enrichment of Scp160p at the ER (17). scp160
mutants are
viable, but display defects in cell morphology and nuclear segregation,
resulting in cells with increased size and DNA content per cell (17,
18). The mechanism by which this complex phenotype is established in
scp160 cells is unknown. In Drosophila, a
functional homologue of Scp160p has also been identified. This protein,
DDP1, binds dodeca satellite repeat regions of centromeric
heterochromatin in embryonic and larval cell nuclei (19, 20).
Overexpression of the DDP1 protein complements the cell morphology and
nuclear segregation defects in
scp160 mutants (19).
Similar multi KH-domain proteins are found ubiquitously in all
eukaryotic cells. In vertebrate species this protein is known as
vigilin (21-23). A clear picture of the cellular function and of
specific RNA targets of these proteins has not yet emerged.
Northwestern blot analyses with Scp160p demonstrated RNA binding
activity in vitro with low sequence specificity (24). RNA
gel mobility shift assays and mRNA affinity column chromatography
showed that Xenopus vigilin binds specifically to the
3'-untranslated region of vitellogenin mRNA (23, 25), stabilizing
the RNA by blocking cleavage by an endonuclease (26). In contrast to
these results, Kruse et al. (27, 28) propose that human
vigilin may be involved in binding and transport of tRNA.
Recently, Scp160p was reported to be associated with cytosolic
polysomes as a component of an messenger RNP complex containing poly(A)-binding protein, which is released upon EDTA treatment (29).
These observations are contradictory to the previously reported
localization of Scp160p predominantly at the ER membrane (17). To
resolve this apparent discrepancy, we decided to investigate the
intracellular distribution of Scp160p more carefully and to ask
specifically whether Scp160p interacts with both cytosolic and
membrane-bound polysomes. We could show that Scp160p is enriched at the ER membrane and is associated with polysomes in a
mRNA-dependent manner. Interestingly, we could
demonstrate that this localization requires intact microtubules.
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EXPERIMENTAL PROCEDURES |
Plasmids and Strains--
To replace SCP160 with an
engineered gene encoding Scp160-GFP, we amplified by PCR a DNA
fragment encoding the SCP160 gene from +2465 to the
stop codon using 5'-CACCGATCCAAAGGCTCA-3' and 5'-CCGctcgagATCTTCTTAAGGATTTCAAAACC-3' primers and a genomic
fragment containing the SCP160 gene (YEP13/6 (17)) as
template. The resulting fragment was cloned first into TOPOII vector
(TOPO TA cloning kit, Invitrogen), re-excised with SacI and
XhoI, and then fused in-frame to a mutant (S65T, V163A) of
GFP and the NUF2 3'-untranslated region in the
non-replicating plasmid pPS1539 (30). The resulting plasmid, pMS356,
was linearized with Tth111I and used for transformation of a
S288c-derived diploid yeast strain. Ura+ transformants were
selected and checked by fluorescence microscopy for expression of
Scp160p-GFP. To confirm the functional replacement, the diploid strain
was sporulated, and Scp160p-GFP-expressing spores were compared with
wild-type spores. Growth of Scp160p-GFP cells at all temperatures was
indistinguishable from wild-type cells. No increase of cell or nuclear
size was observed in Scp160p-GFP cells.
To replace SCP160 with an engineered gene encoding
GFP-SCP160, we cloned a DNA fragment encoding the
SCP160 gene from
625 to +3, amplified by PCR using
5'-CgagctcGGATCCTTCTTTCTCATTCCTTCATTTAATGTTCG-3' and
5'-CactagtCATTGCAGTTATAATGGAAGGAGGGGG-3' primers. The PCR product was
subcloned in pBlueScript SK+ (Stratagene), resulting in
plasmid pMS403. The URA3 gene was amplified from pRS314 (31)
using 5'-GccactccactggGTACTGAGAGTGCACCACGC-3' and
5'-CccagtggagtggGCGGTATTTCACACCGCAGGG-3' primers and ligated into the
unique BstXI site of pMS403 at position
425 of the
SCP160 gene, resulting in pMS404. Into this plasmid we
cloned the SCP160-coding region generated by PCR from
YEP13/6 using 5'-CCatcgatGTCTGAAGAACAAACCGCTATTGAC-3' and
5'-GgtcgacCGATGGAGAATTCAAAATAGATTC-3' as primers. Upstream of the first
ATG of SCP160, we introduced the GFP-coding region, amplified by PCR from pMS356 with primers
5'-GactagtAAAGGAGAAGAACTCTTCACTGGAGTTG-3' and
5'-CatcgatCCTTTGTATAGTTCATCCATGCCATGTG-3', resulting in pMS405. A
linear 4.4-kilobase BamHI-EcoRI fragment was
generated by restriction digest and transformed into a haploid
S288c-derived yeast strain (32). To express GFP-tagged Tub1p, we
linearized pRS303 (31), containing the HIS3 promotor upstream of
GFP-TUB1 (33), with NheI and transformed the DNA into a
haploid S288c-derived yeast strain. A CEN plasmid containing
SCP160 was generated by subcloning a
BamHI-PstI fragment from YEP13/6 into pRS315
(31), resulting in pMS346.
Antibodies and Immunoblotting--
To detect proteins by
immunoblotting procedures, membranes (Protean, Schleicher & Schuell)
were incubated for 2 h at room temperature with antibodies diluted
in blocking buffer (PBS, 0.2% Tween 20, 5% dry milk powder) followed
by peroxidase-conjugated secondary antibodies (Sigma) and detection
with ECL (Roche Molecular Biochemicals). Antibodies to Scp160p were
raised in rabbits (New Zealand White) using glutathione
S-transferase-tagged Scp160p, expressed, and purified from
Escherichia coli as an antigen. Antibodies against ribosomal
proteins Rps3p and Rpl35p were raised in rabbits, using standard
techniques (34), against the peptides VALISKKRKLVADC-CONH2 and CPIRKYAIKV-COOH, respectively, conjugated to keyhole limpet hemocyanin (Pierce) with sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce).
Antiserum specific for Kar2p was produced as described (35). Antibodies
specific to Sec61p have been described previously (36). Antibodies
against Zwf1p were obtained from Sigma. For immunodetection, antibody
dilutions were as follows: Scp160p (1:2,000 to 1:10,000), Rps3p
(1:250,000), Rpl35p (1:25,000), Kar2p (1:10,000), Sec61p (1:10,000),
and Zwf1p (1:5,000).
Cell Lysis and Fractionation--
Yeast cells were grown at
30 °C in complete (yeast extract/peptone/dextrose) or selective
medium (Hartwell Complete media (37)) to
A600 = 0.5-0.6. Cycloheximide was added
to a final concentration of 100 µg/ml, and cultures were grown for
another 15 min at 30 °C. Cells were harvested and resuspended in a
1/100 culture volume of ice-cold low-salt (LS) buffer (20 mM Hepes-KOH, pH 7.6, 100 mM potassium acetate,
5 mM magnesium acetate, 1 mM EDTA, 2 mM dithiothreitol, 100 µg/ml cycloheximide, 0.1 mM phenylmethylsulfonyl fluoride, complete protease
inhibitor mix (according to the manufacturer; Roche Molecular
Biochemicals)). After the addition of two cell volumes of glass beads,
cells were lysed by vigorous shaking for 5 min at 4 °C. Crude
lysates with a protein concentration of 7-12 mg/ml were obtained by
centrifugation for 2 min at 1,200 × g. These lysates
were fractionated by consecutive centrifugation at 6,000, 18,000, and
200,000 × g for 20 min at 4 °C. After each centrifugation step, the pellets were rinsed with ice-cold water and
frozen in liquid nitrogen; supernatants were then subjected to the next
centrifugation step.
Sucrose Density Gradient Fractionation--
Lysates in LS
buffer, LS buffer with additional 7 mM EDTA, LS buffer with
25 units/ml micrococcal S1 nuclease or 500 mM potassium acetate buffer were subjected to centrifugation at 6,000 × g for 20 min. 150 µl of the supernatant (corresponding to
7.5 A600) was loaded on a linear 4-ml 10-40%
(w/v) sucrose gradient in LS buffer or, if lysates were pretreated with
EDTA, in LS buffer with additional 7 mM EDTA. Gradients
were centrifuged at 55,000 rpm in an SW60 rotor (Beckman) at 4 °C
for 1 h and then fractionated into 400-µl fractions using an
ISCO640 gradient fractionator, with continuous monitoring of absorbance
at 254 nm. Each fraction was precipitated with 10% (w/v)
trichloroacetic acid, and the resulting pellet was washed with 80%
(v/v) acetone, dried, and resuspended in SDS sample buffer.
Microscopy--
Cells expressing GFP fusion proteins grown to
A600 = 0.3-0.6 in selective media at 25 °C
were observed under an Olympus BX60 fluorescence microscope with a
Zeiss UPlanAPO 100× oil immersion objective. A Hamamatsu C4742-95 CCD
camera and Openlab software package (Improvision, Heidelberg, Germany)
were used to acquire pictures. Exposure times varied corresponding to
the expression level of the GFP fusion protein from 0.5 s
(Sec63-GFP) to 3 s (GFP-Scp160p). To analyze the actin
cytoskeleton, cells were grown to 0.6 A600 and
fixed with 3.7% (w/v) formaldehyde for 1 h. Spheroplasts were
prepared and transferred onto slides coated with 0.02% (w/v) poly-lysine. After a 5-min treatment with PBS, 0.1% (w/v) Tween 20 and
30 min with PBS, 1% (w/v) bovine serum albumin, spheroplasts were incubated for 40 min in 0.66 µM (20 units/ml)
AlexaTM488-phalloidin (Molecular Probes) in PBS, 1% bovine
serum albumin followed by 0.1 µg/ml
4'6-diamido-2-phenylindoledihydrochloride (DAPI) in PBS for 5 min.
Cells were washed in PBS and mounted with PBS in 80% (v/v) glycerol.
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RESULTS |
Scp160p Cofractionates with Free and Membrane-bound
Polysomes--
Scp160p was previously reported to be associated with
cytosolic polysomes (28). However, indirect immunofluorescence revealed that the majority of Scp160p is localized at the ER membrane (17), and
this large fraction was not analyzed with respect to polysome association. Therefore, we decided to investigate the intracellular distribution of Scp160p by whole cell fractionation to clarify whether
Scp160p is predominantly associated with free or membrane-bound ribosomes. To analyze the entire cellular pool of Scp160p, we generated
a total lysate from cycloheximide-treated wild-type yeast cells by
glass bead lysis in LS buffer containing 100 mM potassium
acetate. From this lysate, we obtained, by consecutive centrifugation
fractions enriched in ER membranes (P6), ribosomes (P18 and P200) and a
ribosome-free cytosol (S200). As shown in Fig.
1, half of the luminal Kar2p was present
in P6, and the other half was found in S200, due to the release of
Kar2p from the ER as a result of mechanical damage to the microsomes by
glass bead lysis. Almost all of the ER membrane marker Sec61p was found
in P6, with a minor fraction in P18. The cytosolic marker protein glucose-6-phosphate-dehydrogenase (Zwf1p) was only present in S200. As
revealed by immunodetection of the small and large ribosomal proteins
Rps3p and Rpl35p, respectively, about half of all ribosomes was found
in the P200 fraction. A smaller proportion was found in P6 and P18.
Taken together, this indicates that P6 is enriched in ER membranes, and
P200 is enriched in membrane-free (cytosolic) ribosomes. P18 contains a
small amount of ER and heavy polysomes.

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Fig. 1.
The majority of Scp160p cofractionates with
ER membranes. Total yeast lysate was fractionated by consecutive
centrifugation, resulting in 6,000, 18,000, and 200,000 × g pellets (P6, P18, and P200) and a supernatant (S200).
Pellets and supernatant from 1 A600 units
of yeast cells were separated by 6-16% SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and decorated with
Scp160p-, Sec61p-, and Kar2p-specific antibodies. Ribosomal proteins
and Zwf1p were analyzed in a similar manner from 0.2 A600 units of cells using antibodies recognizing
Rps3p of the small ribosomal subunit, Rpl35p of the large ribosomal
subunit, and Zwf1p.
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A large proportion of Scp160p sedimented with the membrane-containing
fraction (Fig. 1); a second, smaller pool was found in the cytosolic
ribosome fraction (P18 and P200). Ribosome-depleted cytosol (S200) was
almost free of Scp160p. Therefore, Scp160p is clearly enriched in ER
membrane-containing fractions.
Next we analyzed the association of Scp160p with cytosolic ribosomes by
density gradient centrifugation on linear 10-40% sucrose gradients.
All Scp160p present in the membrane-depleted fraction comigrated with
polysomes (Fig. 2A).
Scp160p-containing complexes smaller than monosomes could not be
detected. Additional density centrifugation with gradients from 10 to
50% sucrose showed an accumulation of Scp160p in fractions containing
heavy polysomes (data not shown). In our analysis, the comigration of
Scp160p with cytosolic polysomes was very labile. Complete comigration of Scp160p with polysomes was only observed in lysates processed immediately. In lysates from frozen cells, the amount of polysomes was
reduced, and some slower migrating Scp160p complexes appeared (data not
shown). The cofractionation of Scp160p with polysomes is
salt-sensitive, indicating an ionic interaction between Scp160p and
ribosomes. After treatment with 500 mM potassium acetate, most Scp160p was found in the ribosome-free fractions on top of the
gradient (Fig. 2B). Removal of Mg2+ ions by the
addition of EDTA leads to the dissociation of 80 S ribosomes and
polysomes into partially disassembled and unfolded ribosomal subunits
and the release of mRNA and 5 S RNA (38, 39). After EDTA treatment,
all Scp160p was shifted into a slower sedimenting complex (Fig.
2C, fractions 1-5), which peaked in fractions
2-4, suggesting that EDTA treatment causes the
release of a Scp160p-containing complex that sediments at similar
position as the 40 S ribosomal subunit. A similar sized messenger RNP
particle, which contains Scp160p and is formed in the presence of EDTA
has been described previously (28).

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Fig. 2.
The membrane-free fraction of Scp160p binds
polysomes. Lysate from cycloheximide-treated yeast cells was
divided, and aliquots corresponding to 7.5 A600
units of cells were incubated for 15 min at 23 °C in LS
buffer (A), in buffer with 500 mM potassium
acetate (KOAc; B), or in LS buffer with 8 mM EDTA (C). To digest mRNA, one aliquot was
treated for 15 min with 25 units/ml micrococcal S1 nuclease in the
presence of 2 mM CaCl2 (D). Nuclease
digestion was stopped by the addition of 3 mM EGTA. Treated
lysates were centrifuged for 20 min at 6,000 × g, and
supernatants were subjected to sucrose density centrifugation (10-40%
sucrose). Gradients were fractionated from the top (fraction
1) to bottom (fraction 10), and the distribution
of Scp160p, Rps3p, and Rpl35p was analyzed by immunodetection. The
state of ribosomes was monitored by the rRNA profiles measured at 254 nm and is shown in arbitrary units.
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To distinguish whether mRNA contributes to the association of
Scp160p with polysomes or whether Scp160p interacts directly with naked
ribosomes, we selectively digested mRNA by mild treatment with
micrococcal S1 nuclease. After mRNA digestion, ribosomes migrated
as monosomes in the sucrose gradient (Fig. 2D, fractions 6-9). The vast majority of Scp160p was found in fractions
1 and 2; smaller amounts comigrated with
ribosomal subunits and monosomes (Fig. 2D, fractions
3-7), indicating that the association of Scp160p with
ribosomes requires the presence of mRNA. Treatment with higher concentrations of S1 nuclease caused an additional shift of Scp160p into the top fractions of the gradient (data not shown). The increased amount of fractionated rRNA seen after EDTA and S1 nuclease treatment (Fig. 2, C and D) corresponds to disassembled
heavy polysomes that pellet in the absence of these agents.
To investigate whether Scp160p from the membrane-enriched fraction (P6)
also interacts with polysomes, we resuspended P6 in LS buffer
containing the nonionic detergent Nikkol and separated the
solubilized material from the unsolubilized fraction by
recentrifugation at 6,000 × g. Under low salt
conditions, half of Scp160p and more than half of the Sec61p and
ribosomes were solubilized (Fig.
3A). Due to the low salt
concentration present during the Nikkol treatment, the solubilization
of membranes was not complete.

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Fig. 3.
Scp160p binds to ER membranes.
A, the membrane-containing fraction was resuspended in LS
buffer (lanes 1 and 2) or LS buffer containing
2% Nikkol (lanes 3-8) and incubated for 15 min at
23 °C. Samples were centrifuged again (20 min; 6,000 × g) to separate unsolubilized (lanes 1 and
3) from solubilized material (lanes 2 and
4). The solubilized material was centrifuged again for 20 min at 6,000 × g (lanes 5 and 6)
or 200,000 × g (lanes 7 and 8).
The distribution of Scp160p, Kar2p, Sec61p, and ribosomes was analyzed
by immunodetection. B, solubilized P6 was centrifuged for 20 min at 6,000 × g, and the supernatant was subjected to
10-40% sucrose density gradient centrifugation. The distribution of
ribosomes, Scp160p, and Sec61p was analyzed by immunodetection. The
state of ribosomes was monitored by the A254
profile.
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Solubilized Scp160p cosedimented with ribosomes at high speed
centrifugation (20 min; 200,000 × g; Fig.
3A, lanes 7 and 8), suggesting that
the Scp160p present at the ER membrane is also bound to polysomes. To
confirm this, we subjected Nikkol-solubilized P6 to sucrose density
gradient centrifugation. Solubilized ribosomes migrated as a major peak
containing monosomes (Fig. 3B, fraction 6) and as
smaller peaks containing polysomes (Fig. 3B, fractions 7-10). The vast majority of Sec61p cofractionated with
ribosomes, suggesting that the binding of ribosomes to the Sec61p
complex is maintained. Some of the Scp160p migrated in fractions 2-5; however, the majority of Scp160p remained in the mono- and
polysome-containing fractions (Fig. 3B, fractions
6-10). This indicates that Scp160p associates with
membrane-bound polysomes.
Dissociation of Ribosomes or Digestion of mRNA Releases Scp160p
from the Rough ER--
To compare the interaction of Scp160p with
cytosolic polysomes and membrane-bound ribosomes, we treated P6 either
with high salt or EDTA or digested the mRNA. A control incubation
for 15 min at 23 °C in LS buffer did not release a significant
amount of Scp160p from P6. Incubation with 500 mM potassium
acetate or removal of Mg2+ ions by EDTA led to the
extraction of more than two-thirds of the Scp160p from P6 (Fig.
4A); similar to the results
with cytosolic ribosomes (Fig. 2B). This indicates that 500 mM potassium acetate abolished the interaction of Scp160p
with membrane-bound ribosomes. EDTA treatment released Scp160p and
ribosomes from the membrane, indicating again a ribosome-mediated
binding of Scp160p to the ER membrane. Digestion of mRNA by S1
nuclease treatment led to the release of some Scp160p. However,
compared with the incubation with EDTA, the release of Scp160p and
ribosomes was less efficient. The release of the nascent chain from
translating ribosomes by puromycin/GTP at low salt did not cause the
release of additional Scp160p from P6. To investigate whether the
interaction of Scp160p with solubilized membrane ribosomes is
mRNA-dependent, we solubilized P6 from untreated lysate
and from S1 nuclease-treated lysate and analyzed the material by
density gradient centrifugation. Half of Scp160p from the solubilized,
untreated P6 fraction comigrated with the ribosome peaks (Fig.
4B, fraction 5-10). Digestion of mRNA
reduced the amount of membrane-bound ribosomes that stay attached to
the membrane (compare ribosome peak in fraction 5 and
6; Fig. 4, B and C). Ribosomes that
were not directly associated with the Sec61p complex or with other
proteins of the ER membrane were removed. Digestion of mRNA also
abolished the cofractionation of Scp160p with ribosomes (Fig.
4C). The vast majority of Scp160p was found in fractions
1 and 2, indicating that this association of
Scp160p with membrane-bound ribosomes is dependent on mRNA.

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Fig. 4.
Scp160p interacts with membrane-bound
ribosomes. A, the membrane-containing fraction (P6) was
incubated at 23 °C for 15 min in LS buffer (mock) in
buffer with 500 mM potassium acetate (HS), in LS
buffer with 8 mM EDTA (EDTA), in LS buffer with
25 units/ml micrococcal S1 nuclease (S1), or in LS buffer
with 1 mM puromycin and 2 mM GTP
(puromycin). Samples were centrifuged again (20 min;
6,000 × g) to reisolate the membrane-containing
fractions. Distribution of Scp160p, Kar2p, Sec61p, and ribosomes after
the indicated treatment was analyzed by immunodetection. B
and C, P6 isolated after mock treatment (B) or
treatment with S1 nuclease (C) was resuspended in LS buffer
containing 2% Nikkol and incubated at 23 °C for 15 min.
Solubilized material was separated by centrifugation (20 min;
6,000 × g) and subjected to sucrose density gradient
centrifugation. The distribution of ribosomal proteins and Scp160p was
assayed as in Fig. 3.
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GFP-tagged Scp160p Is Enriched at the ER of Living Yeast
Cells--
To investigate the dynamic distribution of Scp160p in
living cells, gene fusions of SCP160 were created that
encode the entire Scp160p fused to a bright derivative of GFP (40). The
GFP moiety was fused either at the N or C terminus of Scp160p. These
constructs were then introduced into the yeast genome to completely
replace wild-type Scp160p, making the GFP fusion protein the only
functional copy in the cell. This approach allowed us to study the
distribution of Scp160p at its normal expression levels.
An immunoblot probed with anti-Scp160p antibodies confirmed that the
GFP fusions are the only version of Scp160p present in the cells (Fig.
5A). Furthermore, the levels
of the tagged forms of Scp160p present in the cells were similar to
that of untagged Scp160p in wild-type cells. Fluorescence microscopy
showed that GFP-tagged Scp160p is concentrated around the nucleus and
in patches close to the periphery of the cell (Fig. 5C),
reminiscent of ER staining. To correlate this more directly, we
transformed cells with a plasmid encoding a GFP-tagged version of the
ER resident protein, Sec63p. Sec63p-GFP fluorescence showed a broadly
similar pattern to GFP-tagged Scp160p (Fig. 5B). However,
compared with Sec63p-GFP, more GFP-Scp160p signal was seen at regions
typical for cortical ER. GFP-tagged Scp160p and Sec63p-GFP seem to be excluded from the nucleus, but some diffuse cytoplasmic staining of
GFP-tagged Scp160p was also visible. The partial overlapping localization of Scp160p with the integral ER membrane protein Sec63p
confirmed the localization of Scp160p at the ER membrane.

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Fig. 5.
GFP-tagged Scp160p is localized at the
ER. A, equal amounts of lysates from 0.2 A600 wild-type cells (lane 1) or
cells where SCP160 is replaced by SCP160-GFP
(lane 2) or GFP-SCP160 (lane 3) were
separated by SDS-polyacrylamide gel electrophoresis, and the expression
of Scp160p and Scp160p-GFP fusions was analyzed by immunoblotting using
Scp160p-specific antibodies. Rps3p-specific antibodies were used as a
loading control. B, fluorescence microscopy of cells
transformed with a plasmid encoding Sec63p-GFP. C,
fluorescence microscopy of cells where SCP160 is replaced by
GFP-SCP160 or SCP160-GFP.
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Limited Binding Sites for Scp160p at the ER Membrane--
Next, we
localized GFP-tagged Scp160p in cells overexpressing wild-type Scp160p
from a low copy plasmid. Quantitation from immunoblotting indicated an
approximately 4-fold overexpression (data not shown). The increase in
Scp160p levels induced a significant redistribution of GFP-Scp160p from
the ER to the cytosol (Fig. 6A), consistent with a limited
number of binding sites. To investigate whether GFP-Scp160p behaves
similarly as the endogenous Scp160p, we prepared a 6,000 × g supernatant, treated the pellet with Nikkol, and subjected
both fractions to density centrifugation. GFP-Scp160p present in the
supernatant derived from cells transformed with an empty CEN
plasmid cofractionated preferentially with mono- and
polysome-containing fractions (Fig. 6B, top
panel, fractions 6-10). This indicates that the
GFP-tagged Scp160p behaves similarly with respect to its binding to
polysomes as the endogenous Scp160p (Fig. 2). The distribution of
GFP-Scp160p changed dramatically in cells overexpressing Scp160p;
GFP-Scp160p was shifted from the polysome-containing fractions into
fractions 2-4 (Fig. 6B, bottom
panel). This complex, which emerged only at a high concentration of Scp160p, has a similar size as the complex generated by EDTA treatment (Fig. 2). The majority of the membrane-bound GFP-Scp160p cofractionated with ribosomes (Fig. 6C, top
panel, fractions 6-10) and behaved similar to the
endogenous Scp160p (Fig. 3B). Overexpression of Scp160p
abolished the cofractionation of GFP-Scp160p and membrane-bound ribosomes. Increased levels of Scp160p caused the accumulation of a
ribosome-free Scp160p pool (Fig. 6C, bottom
panel, fractions 2-4). The total amount of Scp160p
bound to ribosomes remained roughly constant (Fig. 6, B and
C, bottom panel, fractions 6-10). Taken together, these results suggest a specific, saturable binding of
Scp160p-ribosome complexes at the ER membrane.

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Fig. 6.
Limited binding sites for Scp160p at the ER
membrane. A, localization of GFP-Scp160p in wild-type
and Scp160p-overexpressing cells. Cells transformed with an empty
CEN plasmid (left panel) or a
SCP160-containing CEN plasmid (right
panel) were grown to 0.3 A600 at 25 °C
in selective media and analyzed by fluorescence microscopy.
B, yeast lysates from a wild-type strain and a strain
overexpressing Scp160p were centrifuged for 20 min at 6,000 × g. Supernatants were subjected to sucrose density
centrifugation. The distribution of ribosomes, Scp160p, and GFP-Scp160p
was analyzed by measuring the A254 and
immunodetection with Scp160p-specific antibodies. C, P6 from
a wild-type strain and a strain overexpressing Scp160p was solubilized
in LS buffer containing 2% Nikkol and recentrifuged for 20 min at
6,000 × g. Supernatants were subjected to sucrose
density centrifugation. The distribution of ribosomal proteins,
Scp160p, and GFP-Scp160p was analyzed as in B.
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Microtubule-depolymerizing Drugs Abolish Scp160p Accumulation at
the ER--
The majority of Scp160p-ribosome complexes were found at
the ER membrane. To analyze how these complexes get to the ER, we localized GFP-Scp160p in cells where the cytoskeleton was disassembled. We depolymerized the actin network or the microtubules by treatment with the drugs latrunculin A (41) and benomyl (42), respectively. Treatment with 30 µg/ml latrunculin A, which induces a rapid
depolymerization of actin (Fig.
7A, panels d and
h), did not change the localization of GFP-tagged Scp160p
(Fig. 7A, panels a and e). In
contrast, depolymerization of microtubules by benomyl induced a
complete redistribution of GFP-Scp160p from the ER to the cytosol
within 15 min (Fig. 7B, panels a and
f). Complete depolymerization of microtubules after benomyl
treatment is shown by localization of GFP-Tub1p. Spindle
depolymerization was first visible after 15 min and was complete after
2 h; all cells were arrested with dot-like GFP-tubulin
fluorescence (Fig. 7B, panel k). Treatment with
nocodazol, a drug also known to depolymerize microtubules, induced a
similar redistribution of GFP-Scp160p (data not shown). The perinuclear
staining of Sec63-GFP after treatment with either benomyl or
latrunculin A indicated that the ER structure was not affected (Fig. 7,
A, panels c and g, and B,
panels d and i). The loss of ER localization
after benomyl treatment only occurred in the presence of ongoing
protein synthesis. Elongation arrest induced by cycloheximide abolished
the benomyl-dependent redistribution of Scp160p from the ER
into the cytosol (Fig. 7B, panels b and g). In the absence of benomyl, active translation was not
required to accumulate GFP-Scp160p at the ER, as cells incubated in
cycloheximide showed a similar concentration of Scp160p at the
perinuclear and cortical ER as control cells (Fig. 7B,
panel c and h). Taken together, these data
suggest that (i) the accumulation of Scp160p at the ER depends on
microtubules, (ii) translation is required to release Scp160p from the
ER to the cytosol, and (iii) the anchoring of Scp160p at the ER is
independent of microtubules. Therefore, we propose a
microtubule-dependent transport of Scp160p-ribosome complexes to the ER.

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Fig. 7.
ER localization of Scp160p requires
microtubules. A, localization of GFP-tagged Scp160p
(panels a, b, e, and f),
Sec63p-GFP (panels c and g), and actin
(panels d and h) before and after treatment with
latrunculin A. Cells were grown in selective Hartwell Complete
media to 0.5 A600 units at 25 °C and assayed
immediately before (panels a-c) and after a 15-min
incubation with 30 µg/ml latrunculin A (10 mg/ml stock solution in
Me2SO (DMSO); panels e and
g) or equal amounts of Me2SO (panel
f). For analysis of actin cytoskeleton, cells were treated as
described under "Experimental Procedures." B, cells
expressing GFP-Scp160p (panels a-c), Sec63p-GFP
(panel d), or GFP-Tub1p (panel e) were grown to
0.5 A600 at 25 °C in selective media and
subjected to fluorescence microscopy without treatment. Cells from the
same cultures were treated with 30 µg/ml benomyl (panels f
and k), 30 µg/ml benomyl plus 100 µg/ml cycloheximide
(panel h), or 100 µg/ml cycloheximide (panel g)
for 15 min (panels f-i) or 120 min (panel k) and
immediately subjected to microscopy.
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DISCUSSION |
Scp160p Bound to Polysomes Accumulates at the ER--
Here, we
report that the majority of the multi KH-domain protein Scp160p is
located at the ER, as indicated by fluorescence microscopy in living
cells with functional, GFP-tagged Scp160p and by cell fractionation.
This distribution is consistent with the results of Wintersberger
et al. (17), who show by indirect immunofluorescence that
Scp160p colocalized with the ER marker Kar2p. Our cell fractionation
experiments clearly demonstrate that this large pool of membrane-bound
Scp160p is associated with polysomes. We could also demonstrate that
the minor cytosolic fraction of Scp160p is similarly associated with
polysomes, consistent with a previous report (29). Like these authors,
we could show a salt-sensitive interaction of Scp160p with ribosomes.
Importantly, we now show in addition that this interaction is dependent
upon mRNA and is saturable. EDTA treatment released Scp160p from
polysomes as a complex partially comigrating with, but distinct from,
40 S subunits. This complex contains mRNA (data not shown) and
shares characteristics of the previously described messenger RNP
complex, which was released from cytosolic polysomes upon EDTA
treatment (29).
The cofractionation of Scp160p with large polysomes implies that
Scp160p may either associate with actively translating ribosomes or
with mRNAs carrying stalled polysomes. Binding of Scp160p to the
ribosome is sensitive to low concentrations of S1 nuclease, which
degrades mRNA but leaves rRNA largely intact. This suggests that
the specificity of ribosome binding is dependent on the interaction of
Scp160p with defined mRNAs and not on the interaction of KH domains
with rRNA per se. This is consistent with the fact that binding of Scp160p to polysomes becomes saturated upon mild
overexpression (Fig. 6) where the concentration of Scp160p is still
substoichiometric to the concentration of ribosomes (data not shown).
The localization of Scp160p by fluorescence microscopy showed a clear
concentration of GFP-tagged Scp160p at the ER. Complexes containing
Scp160p, ribosomes, and mRNA could be solubilized from membranes.
Furthermore, ribosomes were required to maintain the association of
Scp160p with the ER, as suggested by the EDTA-dependent release of Scp160p from the membrane fraction. Interestingly, mRNA
digestion only partially released Scp160p from the membrane, although
sucrose density gradient analysis after mRNA digestion and
solubilization showed that Scp160p was no longer associated with
ribosomes. This suggests that other membrane-bound factors are involved
in the association of Scp160p with the membrane.
The accumulation of GFP-tagged Scp160p at the ER diminished upon
overexpression of untagged Scp160p. One possible explanation is a
requirement of specific mRNAs for the targeting of Scp160p ribosome
complexes to the ER. Alternatively, a cytosolic factor required for
binding of Scp160p at the membrane may be limiting.
Overexpressed Scp160p was found in particles with a similar size as the
complex, which was released from polysomes upon EDTA treatment (29).
The functional relevance of these complexes remains obscure; we do not
even know whether such complexes exist in a normal growing yeast cells.
Accumulation of Scp160p at the ER Requires Microtubules--
Most
interestingly, we found a microtubule-dependent
localization of Scp160p-bound polysomes. Until now, it was hypothesized that smaller cells like yeast rely only on the actin network to transport and/or anchor localized mRNAs (43, 44).
Microtubule-dependent long distance transport is known for
large cells such as neurons, oligodendrocytes, and oocytes. For
example, Staufen mediates microtubule-dependent localization and translational control of Oskar mRNA at the
posterior of Drosophila oocytes (45, 46) and anchoring of
Bicoid mRNA at the anterior of the egg (47). Microtubules are
important for the transient interaction of mRNA with the
cytoskeleton during the transport phase as well as for the attachment
of mRNAs at their final destinations. Our findings suggest that
cytosolic Scp160p-ribosome complexes are transported to the ER membrane dependent on intact microtubules. The high steady state concentration of Scp160p at the ER reflects the final destination of such localized complexes. Few examples of mRNA transport to the ER are known. Recently, mammalian Staufen, which is expressed in most tissues, was
implicated in the transport of large RNPs to the ER. Staufen interacts
with microtubules and polysomes and colocalizes with markers of the
rough ER (48-50). However, it remains unclear whether the observed
Staufen-containing particles represent vesiculated ER-Staufen complexes
or whether these complexes themselves are involved in mRNA
transport. The zipcode-binding protein expressed in Xenopus
oocytes promotes microtubule-dependent transport of Vg1
mRNA to a subcompartment of the ER (51). Another example exists in
plant cells, where mRNAs encoding rice seed storage proteins are
targeted to a subdomain of the ER, known as prolamin protein bodies
(52).
Intracellular Targeting of Scp160p-bound Polysomes--
The large
number of RNA binding domains in Scp160p (14 KH domains) provides
different surfaces to form simultaneous contacts with mRNAs and
rRNAs, allowing selective binding to polysomes. Since Scp160p is
associated with polysomes, it is conceivable that the protein plays a
dual role: (i) positioning specific mRNAs at the ER and (ii)
regulating their translation at this site. In summary, our data show a
mRNA-dependent association of Scp160p with
membrane-bound polysomes and microtubule-dependent
accumulation of those complexes at the ER. These results encourage us
to speculate that Scp160p may function as an RNA binding platform
involved in the targeting of a subset of mRNAs to the ER.
Identification of specific mRNA substrates will help us to further
define the cellular function of Scp160p.