Current Biology 16, 15381543, August 8, 2006 2006 Elsevier Ltd All rights reserved
025
Coordination of Endoplasmic
Reticulum and mRNA Localization
to the Yeast Bud
Maria Schmid,1,2 Andreas Jaedicke,1,2,3 Tung-Gia Du,1
and Ralf-Peter Jansen1,*
1
Department of Chemistry and Biochemistry
Gene Center
Ludwig-Maximilians-University Munich
D-81377 Munich
Germany
Summary
Localization of messenger RNAs and local protein
synthesis contribute to asymmetric protein distribu-
tion not only of cytoplasmic but also of membrane or
secreted proteins [1, 2]. Since synthesis of the latter
protein classes occurs at the rough endoplasmic retic-
ulum (ER), mRNA localization and distribution of ER
should be coordinated. However, this coordination is
not yet understood. In yeast, mRNA localization to
the growing bud depends on the myosin Myo4p, its
adaptor She3p, and the specific RNA binding protein
She2p [3]. These proteins mediate the localization of
23 mRNAs including ASH1 mRNA and mRNAs encod-
ing membrane proteins [4]. In addition, Myo4p and
She3p are required for segregation of cortical ER to
the bud [5]. Here we show, with ASH1 mRNA as a model
mRNA, that localizing messenger ribonucleoprotein
(mRNP) particles comigrate with tubular ER structures
to the bud, which requires the RNA binding protein
She2p. Coordinated movement of the ASH1 mRNP
with ER tubules but not their association with each
other depends on Myo4p and She3p. Subcellular frac-
tionation experiments demonstrate a cosegregation of
ER and She2p, which is independent of Myo4p, She3p,
or polysomes. Our findings suggest a novel model for
mRNA localization that involves association of She2p
and mRNPs with ER tubules and myosin-dependent
cotransport of tubules and localized mRNPs.
Results and Discussion
Two functionally related subclasses of ER, perinuclear
and cortical ER, exist in budding yeast, connected by tu-
bules. The tubular structures that emanate from perinu-
clear ER and whose translocation from mother to bud de-
pends on the unconventional myosin Myo4p apparently
represent precursors for the cortical ER [5]. In order to
analyze whether transport of tubular ER structures and
of cytoplasmic mRNPs occur independently or in a coor-
dinated way, we simultaneously followed mRNP and ER
trafficking. ER tubules were visualized by means of a con-
stitutively expressed fusion of GFP with Hmg1p (hydrox-
ymethylglutaryl-coenzyme A reductase), an ER-resident
*Correspondence: rjansen@lmb.uni-muenchen.de
2
These authors contributed equally to this work.
3
Present address: EMBL Heidelberg, Meyerhofstr. 1, D-69115 Hei-
delberg, Germany.
DOI 10.1016/j.cub.2006.06.
Report
enzyme that is required for ergosterol synthesis. The fu-
sion protein is present in perinuclear and cortical ER as
well as in motile ER tubules [5]. ASH1 mRNA containing
six MS2 binding sites in its 30 -untranslated region was
expressed from the GAL1 promoter and visualized by
coexpressed MS2 coat protein (MS2-CP) fused to the
RedStar fluorescence protein (see Supplemental Data
available with this article online). The MS2-CP-RedStar
fusion contains a nuclear localization signal to target
unbound protein to the nucleus in order to allow better
visualization of cytoplasmic RNP particles recognized
by MS2-CP [6]. Although ASH1 mRNA is usually tran-
scribed only during mitosis when buds have reached
their mature size, we chose this mRNA as model RNA
because it can be effectively localized to the bud at any
stage of the cell cycle [7]. In order to minimize bleaching
effects and allow for prolonged serial image acquisition,
single-focal-plane images were taken every 20 s.
In accordance with previous observations [5, 8], ER tu-
bules move from mother cells into small- or medium-
sized buds. Larger buds (with a volume between 10%
and 25% of the mother cell) were excluded from the anal-
ysis because they already contained tubular and cortical
ER structures. In the cells analyzed, we observed a co-
localization of ASH1-MS2 RNP particles with tubular
ER structures in the bud and in the mother cell (Figure 1,
Movie S1). Particles were visible decorating ER tubules
along the entire length, but frequently a particle was
found at the tip of a moving ER tubule (Figures 1A and
1B, Movie S2). Colocalization of mRNP particles and
ER tubules was detectable over time spans up to 5 min
(Figure 1C, Movies S1 and S2). This suggests that ER tu-
bules and ASH1-MS2 mRNPs move in a coordinated
manner.
In order to test whether the Myo4p/She3p motor pro-
tein complex is needed for the association of mRNPs
and ER tubules, we examined the colocalization in cells
lacking Myo4p. In more than 80% of myo4D cells ob-
served, ER tubules and mRNPs do not move into
small-sized buds. In the remaining cells (<20%), we de-
tected ER tubules in the bud that are not associated
with ASH1-MS2 mRNPs (Figure 1D). This indicates that
a fraction of ER tubules is able to move into the bud inde-
pendently of Myo4p and is consistent with a study that
suggested that Myo4p-dependent transport might not
be the sole mechanism for movement of ER tubules
into the bud [9]. Strikingly, tubules remaining in the
mother cell were still associated with mRNP particles,
and both tubules and mRNP particles showed coordi-
nated yet random movement (Figure 1D, Movie S3), sug-
gesting that colocalization of ASH1-MS2 mRNPs and ER
tubules is independent of the Myo4/She3p complex. We
next investigated the role of the RNA binding protein
She2p. In more than 50% of she2D cells imaged, only
smaller, less bright particles were observed, possibly
due to formation of less stable or smaller mRNPs in the
absence of She2p [6]. In the remaining cells, the ASH1-
MS2 mRNP particle stays in close proximity to the
Cosegregation of ER and Localized mRNA
1539
nuclear envelope or perinuclear ER (Figure 1E, Movie
S4). ER tubules emanating from perinuclear ER in
she2D cells are not associated with RNP particles, which
is in contrast to wild-type cells where a dynamic comi-
gration of cytoplasmic ER tubules and an ASH1-MS2
RNP can be observed (Figure 1C, Movie S1). Accord-
ingly, in situ hybridization to detect untagged ASH1
mRNA expressed under similar conditions in she2D cells
shows perinuclear staining (Figure S1), ruling out an arti-
ficial defect resulting from the MS2 tagging. These data
suggest that She2p but not Myo4p is required for the as-
sociation of a localizing mRNP with ER tubules that grow
toward the bud.
In order to support the hypothesis of an association of
ER and localizing mRNPs, we determined whether the
She2p protein cofractionates with ER. She2p binds to
all described mRNAs that localize to the bud tip,
Figure 1. Colocalization of ASH1-MS2 mRNP
Particles with Tubular ER in the Bud
(A and B) Representative examples of cells
from strain RJY2339 with ASH1-MS2 mRNP
particles (arrows) labeled by MS2-RedStar
fusion protein and ER tubules (arrowheads)
labeled by Hmg1p-GFP fusion protein.
ASH1-MS2 particles (red) colocalize with the
tip of ER tubules (green) in the bud.
(C) Individual frames from a time-lapse series
(Movie S1) of the cell shown in (A). The ASH1-
MS2 mRNP (red, arrow) stays associated with
an ER tubule (green) for more than 3 min. Ar-
rowhead marks the tip of the ER tubule. Time
point of each image is indicated.
(D) Colocalization of ASH1-MS2 mRNP with
ER tubules in the absence of Myo4p. Individ-
ual images from a time-lapse series (Movie
S3) of strain RJY2372 showing MS2-Red-
Star-labeled ASH1-MS2 RNP particle (red, ar-
row) associated with tubular ER structures
(green, arrowhead) in the mother cell. Note
that in contrast to wild-type cells (C), the
marked tubule does not show directional
movement to the bud and that no ASH1-
MS2 particles are visible in the bud (at the
bottom of the cell).
(E) Colocalization of ASH1-MS2 mRNP with
perinuclear ER in a she2D cell. Individual im-
ages from a time-lapse series (Movie S4) of
strain RJY2377 showing an MS2-RedStar-la-
beled ASH1-MS2 mRNP particle (red, arrow)
associated with perinuclear ER. Note that an
ER tubule (green, arrowhead) emanating
from the perinuclear ER is not associated
with an RNP particle.
including ASH1 mRNA [4]. In a first approach, cell ex-
tracts were separated on a linear sucrose gradient and
the distribution of proteins was analyzed by Western
blots (Figure 2A). Hmg1p-GFP migrates close to the bot-
tom of the gradient. Similar migration behavior is seen for
two other ER proteins, Dpm1p (dolichol phosphate man-
nose synthase) and Sec61p, the core component of the
translocon (Figure 3). As shown before, an HA-tagged
version of Myo4p cofractionates with ER marker proteins
(Figure 2A) [5]. Like Myo4p, She2p also fractionates with
an ER marker (Figure 2A). Since She2p can shuttle be-
tween nuclei and cytoplasm [10], we first tested whether
the heavy fractions containing She2p are different from
nuclei. A crude membrane fraction from lysed yeast cells
was separated on a two-step sucrose gradient in order to
distinguish ER microsomes from nuclei. Whereas ER
membranes accumulate at the interphase of the two
Current Biology
1540
Figure 2. The ASH1 mRNA Binding Protein She2p Cofractionates
with Rough Endoplasmic Reticulum
(A) Sucrose velocity gradient centrifugation. Cell extract from strain
RJY2479 (MYO4-HA6, URA3::HMG1-GFP) was separated on a linear
18%60% sucrose gradient as described in Supplemental Experi-
mental Procedures. Aliquots of 12 fractions and the pellet were an-
alyzed by Western blotting against HA, GFP, or She2p. She2p and
Myo4p comigrate with the ER marker protein Hmg1p-GFP to the
dense fractions of the gradient (fractions 24).
(B) Purification of yeast ER membranes from a crude membrane pel-
let by centrifugation through a sucrose cushion. Numbers corre-
spond to fractions from top to bottom. F4, the interphase between
1.2 M and 1.5 M sucrose cushions, contains ER. She2p is also en-
riched in F4, but the nuclear marker protein Rpa49p is mainly found
in F1 (broken nuclei) and F7 (nuclei).
(C) Flotation assay. Crude yeast membranes equilibrated in 50%
sucrose were overlaid with two sucrose cushions containing 0% or
40% sucrose. After equilibrium centrifugation, fractions were taken
and analyzed by Western blotting. Numbers on top indicate sucrose
concentrations of the corresponding sucrose cushions or inter-
phases between cushions. She2p and Hmg1p-GFP but not Rpa49p
float to the 0%/40% sucrose interphase, which indicates a mem-
brane association.
sucrose cushions (Figure 2B, lane 4), a marker for soluble
nuclear proteins (Rpa49p, a subunit of RNA polymerase
I) is found at the bottom of the gradient (lane 7) or at the
top (lane 1). Rpa49p in fractions 1 and 2 corresponds
to protein released from broken nuclei, whereas the
Rpa49p signal in the pellet reflects nuclear protein in
intact nuclei. In contrast to Rpa49p, a significant amount
of She2p is present at the cushion interphase, suggest-
ing cofractionation of She2p and ER. In order to rule
out that She2p in heavy fractions reflects very large
RNPs that accidentally comigrate with ER, we performed
flotation experiments as described previously for neuro-
nal RNP complexes [11]. Whereas neuronal RNP marker
proteins did not float with membranes to sucrose
with lower density (interphase between 0% and 40%
sucrose) [11], a significant fraction of She2p floats with
Hmg1p (Figure 2C). Although less She2p is floated than
Hmg1p, this is expected since integral membrane
proteins like Hmg1p are stronger associated with ER
membranes than proteins that are peripheral or loosely
associated. More important, the nuclear marker Rpa49p
does not show flotation, indicating that the behavior of
She2p is specific.
Since double live imaging (Figure 1D) indicated that
mRNP association with ER tubules does not depend on
the motor protein complex, we tested whether cosegre-
gation of She2p and ER is also independent of Myo4p or
its adaptor She3p. Deletion of MYO4 or SHE3 does not
affect migration of She2p into the gradient (Figure 3A).
In myo4D, she3D, or wild-type extracts, She2p comi-
grates with three ER marker proteins (Hmg1p-GFP,
Sec61, and Dpm1p) into high-density fractions (lanes
25). In order to test whether association of ASH1
mRNA and She2p with ER is mediated by ongoing trans-
lation and nascent chain-translocon interaction, we
added 10 mM EDTA to the cell extracts in order to disrupt
ribosomal subunits [12]. EDTA treatment had no effect
on She2p-ER comigration into the gradient (Figure 3B
and Figure S3). ER marker and She2p shifted by two to
three fractions toward the top of the gradient, indicating
that the now-less-dense ER had lost attached ribosomes
but kept She2p. This interpretation is further supported
by the observation that the ribosomal protein Rpl13p
disappeared from higher density fractions after EDTA
treatment. In order to assess the role of RNA binding
in She2p-ER association, we generated a mutant of
She2p (N36S) unable to bind to ASH1 mRNA [13]. When
expressed in yeast, this mutant protein accumulates in
nuclei (data not shown), which is in accordance with pre-
vious results that a deletion of the amino terminus of
She2p leads to loss of RNA binding and entrapment of
the protein in the nucleus [10]. Thus, a She2p mutant de-
ficient in RNA binding cannot be used to directly assess
the RNA dependence of a She2p-ER association.
We conclude from the set of cell fractionation experi-
ments described above that She2p cofractionates with
ER membranes independently of Myo4p, She3p, and
ER-attached ribosomes. Association of She2p with ER
could in principle result from direct binding to ER mem-
branes or via adaptor proteins such as She3p [14, 15]. Al-
though previous results suggest a direct interaction be-
tween She2p and She3p [14], She3p is not required for
She2p association with ER (see Figure 3) and is therefore
unlikely to be the linker to the ER. It is feasible that She2p
attaches to both She3p and to ER independently and
that the trimeric She2p-She3p-Myo4p complex that
has been proposed as the basic device for mRNA local-
ization in yeast [3] is only part of a still more complex
transport machinery that includes ER tubules.
The observed association of She2p with ER is not re-
stricted to yeast RNA localization factors. In Xenopus
laevis oocytes, Vg1 RNA binding protein recognizes sev-
eral localized mRNAs including Vg1 [16], and this protein
Cosegregation of ER and Localized mRNA
1541

Figure 3. She2p Association with ER Does Not Require Myo4p, She3p, or Polysomes
(A) Cell extracts from strains RJY2323 (wild-type), RJY2372 (myo4D), and RJY2475 (she3D) were separated on a linear sucrose gradient as de-
scribed in Supplemental Experimental Procedures, and aliquots of gradient fractions were analyzed after centrifugation by Western blotting.
She2p cosediments with three ER marker proteins (Hmg1p-GFP, Sec61p, and Dpm1p) to high-density fractions (14) independently of MYO4
or SHE3.
(B) Extracts treated with 10 mM EDTA to disassemble polysomes or mock-treated were separated as described above. A shift of ribosomal
protein Rpl13p toward less dense fractions (811) of the gradient verifies successful EDTA-mediated polysome disruption. She2p and Hmg1-
GFP cofractionate in both gradients with the peaks of Hmg1p-GFP and She2p shifting from fractions 13 to fractions 35, indicating a loss of
polysomes from the ER.

colocalizes with ER [17]. Similar observations have been
made for Staufen, a conserved double-stranded RNA
binding protein involved in mRNA localization in various
cell types. In Xenopus oocytes as well as in certain mam-
malian cell types, a fraction of Staufen cofractionates
and colocalizes with rough ER [1820].
Our data suggest that the processes of ER segregation
and RNA localization in yeast are coupled, in contrast to
a previous model where RNA trafficking and ER tubule
movement were suggested to be independent [5]. The
previous model was proposed mainly based on the
observation that in aux1D mutant cells, localization of
IST2 mRNA to the bud can still be detected. AUX1 (Aux-
ilin-like protein 1) was originally identified in a screen for
mutants defective in ER segregation [8]. In order to re-
capitulate these experiments with our experimental
system, we determined the distribution of ER tubules
and ASH1-MS2 mRNPs in aux1D, myo4D, srp101-47ts,
and wild-type cells with small- to medium-sized buds.
SRP101 encodes the a subunit of the signal recognition
particle receptor, a heterodimeric protein in the ER mem-
brane. At the restrictive temperature, strains carrying

Figure 4. Localization of ASH1-MS2 RNP

Particles to the Bud Is Impaired in Mutants
Affecting ER Segregation
(A) Top: Representative images of wild-type
cells (RJY2339) with small- or medium-sized
buds containing ASH1-MS2 mRNPs (left) or
cortical ER and ER tubules (middle) in the
bud. Bottom: Representative images of
aux1D mutant (RJY2794) cells showing ab-
sence of ASH1-MS2 RNPs or cortical ER
from the bud.
(B) Quantitative analysis of ASH1-MS2 RNPs
(white bars) and cortical ER (Hmg1p-GFP;
black bars) localization to small- or medium-
sized buds of wild-type, aux1D, myo4D, and
srp101-47ts cells. 164 cells (wild-type), 243
cells (aux1D), 340 cells (srp101-47), or 101
cells (myo4D) were scored in three indepen-
dent experiments. Error bars indicate the
standard error of the mean (SEM).
Current Biology
1542
a srp101-47ts mutation show similar ER segregation de-
fects like aux1D cells [21]. In wild-type cells, the majority
of buds contain ER tubules and ASH1-MS2 mRNPs
(Figure 4A). In contrast, only 40% of srp101-47ts cells,
27% of aux1D cells, and 14% of myo4D cells show
bud-specific cortical ER staining or ER tubules that
have moved into small- to medium-sized buds (Figure
4B). In accordance with the observed defects in ER seg-
regation, all three mutants also affect ASH1-MS2 mRNP
particle localization to small- or medium-sized buds,
which is more pronounced in myo4D cells (0% buds
with a RNP particle) than in aux1D (RNP particle in 24%
of buds) or srp101-47ts (43% buds with RNP particles)
cells. In addition, we observed that ASH1-MS2 RNP sig-
nals in aux1D mother cells are generally weaker than in
wild-type or myo4D cells, possibly due to a defect in
RNP assembly or particle composition. Our observation
apparently contradicts previous findings [5]. The differ-
ence in the observed effects of aux1D on mRNA localiza-
tion might be due to the use of in situ hybridization in the
previous work [5] in contrast to double live imaging,
which might be more meaningful, as indicated by the
fact that we scored dynamic ER tubule segregation and
RNP distribution in the same cell. Our observations of
a parallel loss of RNP particle and cortical ER localization
in aux1D, myo4D, and srp101-47ts mutants support the
notion that transport of ER tubules and of RNP particles
are coordinated and not independent events.
It is interesting to note that ASH1 encodes a transcrip-
tion factor [22]. Thus, in this case an association of the
mRNA with ER must occur independently of signal se-
quences within the encoded protein. We have not di-
rectly addressed the question of the mechanism of
ASH1 mRNA-ER association, but such interaction has
already been reported [23]. Here, a genome-wide analy-
sis of translated mRNAs demonstrated that ASH1 is
mainly translated on ER bound polysomes. In addition,
several localized mRNAs in yeast code for membrane
or secreted proteins [4]. A coordination of their move-
ment with cortical ER should facilitate local synthesis
of the proteins at the cortical ER in the bud, which has
recently been suggested for the Ist2 protein [24].
Several other reports indicate that ER plays an impor-
tant function in mRNA localization or sorting [25]. In as-
cidian eggs, HrPEM and macho-1 mRNAs bind to and
move with rough ER at the cell cortex [26]. In Xenopus
oocytes, cortical ER is implicated in anchoring of local-
ized mRNAs [27]. In addition, a number of nonlocalizing
mRNAs encoding nonmembrane proteins are found to
partition to ER polysomes [28]. Thus, it is conceivable
that coordinated localization of mRNAs encoding mem-
brane-associated as well as nonmembrane proteins and
of ER is a commonly used cellular mechanism that could
help to locally translate and sort proteins.
Supplemental Data
Supplemental Data include three figures, four movies, one table, and
Supplemental Experimental Procedures and can be found with
this article online at http://www.current-biology.com/cgi/content/
full/16/15/1538/DC1/.
Acknowledgments
We thank M. Seedorf, M. Kiebler, and I. Mattaj for helpful comments
on the manuscript; H. Tschochner and M. Seedorf for antibodies
against Rpa49p, Rpl13p, and Sec61p; R. Hampton and M. Knop for
providing plasmids pRH457 and pSR8; and W. Prinz for strain
srp101-47. R.-P.J., A.J., and T.-G.D. were supported by grants of
the German Research Foundation (DFG JA696/4-3; SFB646 TP A5),
and M.S. was supported by a grant from the Bavarian Elite Network.
Received: March 26, 2006
Revised: May 30, 2006
Accepted: June 6, 2006
Published: August 7, 2006
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