The Hsp40 chaperone Jjj1 is required for the nucleo-cytoplasmic recycling of preribosomal factors in Saccharomyces cerevisiae

Défenses Antivirales et Antitumorales, CNRS-UMR5235, Université Montpellier II, France.
RNA (Impact Factor: 4.94). 10/2007; 13(9):1570-81. DOI: 10.1261/rna.585007
Source: PubMed
ABSTRACT
Ribosome biogenesis is a major conserved cellular pathway that requires both ribosomal proteins and many preribosomal factors. Most of the pre-60S factors are recycled into the nucleus; some of them shuttle between the nucleus and the cytoplasm while a few others, like Rei1, are strictly cytoplasmic and are mostly involved in the dissociation/recycling of the pre-60S shuttling factors. Here, we investigated the role of the Jjj1 Hsp40 chaperone in ribosome biogenesis. The absence of Jjj1 leads to a cold sensitive phenotype, a defect in the relative amount of the large ribosomal subunit with the appearance of halfmers, and to cytoplasmic accumulation of shuttling factors such as Arx1 and Alb1, which stay bound to the pre-60S particles. Jjj1 is, thus, a novel pre-60S factor involved in the last cytoplasmic steps of the large ribosomal subunit biogenesis. We report the biochemical association of Jjj1 and Rei1 to similar pre-60S complexes, their two-hybrid interactions, and their functional links. Altogether, these results indicate that Rei1 and Jjj1 share many common features. However, while the functions of Jjj1 and Rei1 partially overlap, we could distinguish specific role of the two proteins in Arx1/Alb1 and Tif6 recycling. We propose that Jjj1 is preferentially required for the release of Arx1 and Alb1 shuttling factors from the cytoplasmic pre-60S particles while Rei1 is preferentially involved in their recycling.

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Available from: Emilie Demoinet, Jan 05, 2015
The Hsp40 chaperone Jjj1 is required for the
nucleo-cytoplasmic recycling of preribosomal
factors in Saccharomyces cerevisiae
EMILIE DEMOINET,
1,2
ALAIN JACQUIER,
2
GEORGES LUTFALLA,
1
and MICHELINE FROMONT-RACINE
2
1
De
´
fenses Antivirales et Antitumorales, cc86 CNRS-UMR5235, Universite
´
Montpellier II, 34095 Montpellier Cedex 5, France
2
Unite
´
de Ge
´
ne
´
tique des Interactions Macromole
´
culaires, 2171CNRS-URA, Institut Pasteur, F-75724 Paris Cedex 15, France
ABSTRACT
Ribosome biogenesis is a major conserved cellular pathway that requires both ribosomal proteins and many preribosomal
factors. Most of the pre-60S factors are recycled into the nucleus; some of them shuttle between the nucleus and the cytoplasm
while a few others, like Rei1, are strictly cytoplasmic and are mostly involved in the dissociation/recycling of the pre-60S
shuttling factors. Here, we investigated the role of the Jjj1 Hsp40 chaperone in ribosome biogenesis. The absence of Jjj1 leads to
a cold sensitive phenotype, a defect in the relative amount of the large ribosomal subunit with the appearance of halfmers, and
to cytoplasmic accumulation of shuttling factors such as Arx1 and Alb1, which stay bound to the pre-60S particles. Jjj1 is, thus, a
novel pre-60S factor involved in the last cytoplasmic steps of the large ribosomal subunit biogenesis. We report the biochemical
association of Jjj1 and Rei1 to similar pre-60S complexes, their two-hybrid interactions, and their functional links. Altogether,
these results indicate that Rei1 and Jjj1 share many common features. However, while the functions of Jjj1 and Rei1 partially
overlap, we could distinguish specific role of the two proteins in Arx1/Alb1 and Tif6 recycling. We propose that Jjj1 is
preferentially required for the release of Arx1 and Alb1 shuttling factors from the cytoplasmic pre-60S particles while Rei1 is
preferentially involved in their recycling.
Keywords: ribosome biogenesis; pre-60S maturation; chaperone; nucleocytoplasmic transport; pre-ribosomal factors recycling;
yeast Saccharomyces cerevisiae
INTRODUCTION
The ribo some is one of the most important cellular
macromolecular structures in terms of function, size, and
the energy that a cell consumes for its biogenesis (Warner
1999). R ibosome biosynthesis begins with the transcriptio n
of the 35S and 5S rRNA precursors by RNA pol I and III,
respectively. The processing of the 35S rRNA precursor
generates the 18S rRNA (backbone of the small ribosomal
subunit) and the 5.8S and 25S rRNAs (backbone of the
large ribosomal subunit). The rRNAs are embedded in non-
coding spacer regions, the external transcribed sequences,
59- and 39-ETS, and the internal transcribed sequences,
ITS1 and ITS2 (Venema and Tollervey 1999). Association
of the 35S rRNA precursor with ribosomal and preribo-
somal factors generates the large 90S preribosomal complex
that undergoes various steps of maturation such as chem-
ical modifications (Decatur and Fournier 2002) and exo-
and endonucleolytic cleavages (Venema and Tollervey
1999) to remove the ETS and ITS regions. Among a well-
orchestrated series of cleavage events, the A2 proces sing
generates the large 60S precursor particles containing the
27SA2 intermediate rRNA and the small 40S precursor
particles containing the 20S intermediate rRNA (Fatica and
Tollervey 2002; From ont-Racine et al. 2003; Tschochner
and Hurt 2003). Then, both pre-ribosomal precursor
particles follow independent routes; the pre-40S particle
is mostly processed in the cytoplasm, whereas the matura-
tion of the pre-60S particle is mostly achieved in the
nucleus before export to the cytoplasm where final matu-
ration takes place.
In addition to the large ribosomal protein themselves,
around 80 facto rs have now been predicted or shown to
Reprint requests to: Micheline Fromont-Racine, Ge
´
ne
´
tique des Inter-
actions Macromole
´
culaires, Institut Pasteur, 25 rue du docteur Roux,
75724 Paris Cedex 15, France; e-mail: mfromont@pasteur.fr; fax: +33
14568790; or Georges Lutfalla, De
´
fenses Antivirales et Antitumorales, cc86
CNRS-UMR5235, Universite
´
Montpellier II, Place Euge
`
ne Bataillon, 34095
Montpellier Cedex 5, France; e-mail: lutfalla@univ-montp2.fr; fax: +33 467
14 92 01.
Article published online ahead of print. Article and publication date are
at http://www.rnajournal.org/cgi/doi/10.1261/rna.585007.
1570 RNA (2007), 13:1570–1581. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2007 RNA Society.
Page 1
participate in the biogenesis of the large ribosomal subunit
(Fatica and Tollervey 2002; Fromont-Racine et al. 2003;
Tschochner and Hurt 2003). In contrast to the ribosomal
proteins, pre-ribosomal factors associate temporarily with
the maturing subunits. Most of them associate with and
dissociate from the subunits into the nucleus. A few other
factors like Arx1, Alb1, Tif6, Rlp24, and Nmd3 are shuttling
factors (Senger et al. 2001; Nissan et al. 2002; Saveanu et al.
2003; Hedges et al. 2005; Lebreton et al. 2006b). They
bind the pre-60S in the nucleus and di ssociate in the
cytoplasm. Finally, other factors like Lsg1 (Hedges et al.
2005), Rei1 (Hung and Johnson 2006; Lebreton et al.
2006b), Efl1 (Senger et al. 2001), and Drg1 (H. Bergler
and M. Fromont-Racine, unpubl. data) are strictly cyto-
plasmic and required for the release and recycling of
shuttling factors. These final cytoplasmic steps involve
structural rearrangements but no rRNA cleavage. GTPase
or ATPase proteins appear to be involved in this process.
Indeed, Lsg1 GTPase activity is required for Nmd3 release
(Hedges et al. 2005) and in the absence of the Efl1 GTPase,
Tif6 accumulates into the cytoplasm (Senger et al. 2001).
The Drg1 ATPase protein (Zakalskiy et al. 2002) is also
required for the recycling of shuttling pre-60S factors (H.
Bergler and M. Fromont-Racine, unpubl. data). Thus, the
ribosomal cleavage and maturation events are tightly
coupled to nucleocytoplasmic transport. Recycling requires
transport machineries involving karyopherin Kap121,
Kap123, and Kap104, which are used for the import of
shuttling factors (Rout et al. 1997; Leslie et al. 2004) and
exportin (Crm 1/Xpo1) for the translocation of the pre-60S
particles through the nuclear pore (Ho et al. 2000; Gadal
et al. 2001).
In this study, we investigate the role of Jjj1, an Hsp40
chaperone (Meyer et al. 2007) that would be specifically
involved in the biogenesis/maturation of the large ribo-
somal subunit. In eukaryotes, two chaperone networks have
been described, a stress inducible network that protects the
cellular proteome from stress and another stress repressed
chaperone network that is dedicated to protein biogenesis
(Albanese et al. 2006). Besides these ‘‘general’’ chaperones,
some chaperones are specifically involved in structural
transitions for polypeptides in large molecular complexes,
as, for example, auxilin/Hsc70 required for clathrin uncoat-
ing (Ungewickell et al. 1995; Pishvaee et al. 2000; Fotin
et al. 2004). Hsp40s function to specify the cellular targets
of Hsp70 chaperones. In the classical view of the function
of an Hsp40 chaperone, the protein forms a complex with
an unfolded or nonnative protein to prevent its aggregation
or misfolding. Hsp70 proteins recognize short hydrophobic
polypeptide in this complex, but the polypeptide binding is
regulated by the nucleotide bound state. Conformational
changes ge nerated upon ATP hydrolysis stabili ze Hsp70’s
interaction with its polypeptide substrate, whereas the
exchange of ADP for ATP promotes their releas es (Fan
et al. 2003; Hen nessy et al. 2005).
Apart from Jjj1, no chaperone has been found to be
specifically involved in the biogenesis of the large ribosomal
subunit. We show here that Jjj1 is involved in the last steps
of the biogenesis of the ribosomal large subunit and
interacts with Rei1, another late pre-60S factor cytoplasmic
involved in the recycling of the shuttling factors (Arx1,
Alb1). Even if Jjj1 and Rei1 share several common
characteristics, they are required for different maturation
events.
RESULTS
Jjj1 is involved in ribosome biogenesis
We recently demonstrated that Rei1 is involved in the last
cytoplasmic steps of ribosome biogenesis. To gain more
insight into potential partner s of Rei1, a two-hybrid screen
was performed. One of the identified candidates was JJJ1/
YNL227W. In Figure 1A, left panel, the inset coding the Jjj1
protein encompassed a region from amino acid 162 to the
end of the protein. In addition to the two conserved C
2
H
2
Zn fingers from amino acids 338 to 362 and from 549 to
573, Jjj1 contains a N-terminal DNA J domain (up to
amino acid 67) that assigns Jjj1 to the Hsp40 chaperone
family. The DNA J domain was excluded from the selected
Rei1 interacting domain. The physical interaction between
Jjj1 and Rei1 was confirmed by a two-hybrid matrix
approach. To further investigate additional interactions, we
tested physical interactions be tween Jjj1 and several pre-60S
factors previously described as partners of Rei1, such as
Arx1, Alb1, and the ribosomal protein Rpl24. A two-hybrid
interaction was observed between Jjj1 and Rei1 (Fig. 1A,
right panel). No other physical interaction was found with
Jjj1 whereas Rei1 interacted with Rpl24, and Arx1 inter-
acted with itself and with Alb1 (Lebreton et al. 2006b). We
noted that Alb1 was an autoactivating bait. The strongest
two-hybrid signals were obtained between Arx1 and itself
and between Arx1 and Alb1, as demonstrated by the dark
blue color obtained by X-Gal overlay assay.
JJJ1 is not essential in yeast; however, its deletio n leads
to a cold sensitive phenotype. Apart from the DNA J motif,
suggesting a chaperone activity, the function of this protein
was unknown. However, in addition to the physical two-
hybrid links between Rei1 and Jjj1, several lines of evidence
suggested that Jjj1 could be involved in ribosome bio-
genesis. For example, the JJJ1 mRNA level fluctuates
similarly to the levels of mRNAs coding for preribosomal
factors in response to various environmental stresses
(Gasch et al. 2000). Moreover, Jjj1 has been identified in
several preribosomal complexes using the TAP purification
approach (Gavin et al. 2006).
To investigate the involvement of Jjj1 in ribosome
biogenesis, we looked at the polysome profile of a jjj1D
cellular extract from the ED10 (jjj1D) strain after ultracen-
trifugation in a sucrose gradient. The relative amount of
Jjj1 is required for pre-60S factors recycling
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60S subunit was clearly affected in the absence of Jjj1
(Fig. 1B, right panel) as compared to the wild-type strain
(Fig. 1B, left panel). Moreover, we observed a decrease of
the total amount of polysomes and the presence of half-
mers, which reflects the relative deficit in 60S as compared
to 40S during tr anslation initiation. This phenotype was
obvious at 30°C (data not shown) but was even more
pronounced after a shift to 23°C for 2 h (Fig. 1B). To define
FIGURE 1. Jjj1 is involved in the 60S subunit biogenesis. (A) Two-hybrid screen identifies a physical link between Jjj1 and Rei1. (Right panel) Jjj1
was selected as a partner of Rei1 in a two-hybrid genomic screen. The domain of Jjj1 interacting with Rei1 corresponds to amino acids 162–590.
The two putative C
2
H
2
Zn-fingers motifs and the J domain are depicted by black and gray boxes, respectively. (Left panel) Various entire ORF
were cloned into pAS2DD as bait and into pACTIIst as prey vectors, respectively. The bait and the prey vectors were transformed into the CG1945
and Y187 strains, respectively. Both strains were mated and the diploids were selected on minimal medium plates without leucine and tryptophan
(LW). The diploids that display a two-hybrid positive interaction were selected on minimal medium plates without leucine, tryptophan and
histidine (LWH). An X-Gal overlay was performed on the LWH plate. (B) In the absence of Jjj1, the relative amount of 60S subunit is affected.
A wild-type (BY4741) or a jjj1D strain (ED10) was grown in YPGlu and shifted at 23°C for 2 h. The whole-cell extracts from these strains were
separated by ultracentrifugation on a 10%–50% sucrose gradient. Peaks corresponding to the various subunits and polysomes are indicated.
Asterisks note halfmers. (C) Schematic representation of the ribosomal RNAs maturation. The relative position of the oligonucleotides used to
detect specific rRNA by primer extension is indicated. (D) In the absence of Jjj1, the 27SB processing is very weakly affected. Total RNAs were
extracted from culture cells of wild-type strain (BY4741), jjj1D (ED10), and rei1D mutant strains (ED70) in YPGlu medium after a shift to 23°C
for 2 h. Mature and intermediate rRNA were detected either by primer extension (left panel) or by Northern blot on agarose gel (middle panel) or
on acrylamide gel (right panel). The oligonucleotides used are noted. The U2 snRNA was used as a loading control.
Demoinet et al.
1572 RNA, Vol. 13, No. 9
Page 3
which step of ribosome biogenesis is affected by the absence
of Jjj1, we tested the relative amounts of the different
mature and intermediate rRN A in jjj1D strains (ED10 mat
a, ED11 mat a) in comparison with a wild-type (BY4742,
BY4741) or a rei1D (ED70 mat a, ED71 mat a) strain (Fig.
1D). Steady-state rRNA levels were analyzed by primer
extension (Fig. 1D, left panel) and by Northern blotting
either on agarose (Fig. 1D, middle panel) or acrylamide gels
(Fig. 1D, right panel) from wild-type, Jjj1, and Rei1-
depleted cells after a shift to 23°C for 2 h. With the
exception of the 27SB/A2 pre-rRNA ratio, which very slightly
increased (2.9 for the jjj1D strains compared to 1.5 for the
WT strains and 3.2 to rei1D strains based on two experi-
ments, one on each mating type), none of the other ratios
were significantly affected. Similar results were obtained after
a 15-min shift at 23°Cwithrei1D, jjj1D, and WT strains (mat
a, mat a) (data not shown). These results are in agreement
with the decrease of the relative amount of the 60S subunit in
the absence of Jjj1 but suggest that Jjj1 has no direct influence
on nuclear pre-rRNA processing.
Since ribosome maturation mutations are usually corre-
lated with a ribosome export defect, we tested the locali-
zation of the ribosomal Rpl25-GFP fusion protein in the
jjj1D strain. We observed that, like in a rei1D strain or in a
wild-type strain, the fluorescence was mainly located in the
cytoplasm (data not shown), suggesting that the export of
Rpl25-GFP was not affected. In addition, like Rei1, Jjj1 is
a cytoplasmic protein (Huh et al. 2003).
Altogether, these results show that Jjj1
is a cytoplasmic factor affecting nuclear
ribosomal large subunit maturation. This
is reminiscent of the effect described for
rei1D, which participates in the recycling
of shuttling pre-60S factors (Hung and
Johnson 2006; Lebreton et al. 2006b).
Jjj1 physically interacts
with the pre-60S particles
The physical two-hybrid interaction
between Jjj1 and Rei1 correlates with
similar defects of the 60S biogenesis in
the absence of either of these proteins.
To check whether Jjj1, like Rei1, is
biochemically associated with the 60S
particles, we performed a sucrose gra-
dient experiment using a chromosomal
Jjj1-TAP fusion protein (OT52) (Open
Biosystem). After sedimentation of the
whole cellular extracts on sucrose gra-
dient, we observed that Jjj1-TAP fusion
protein was present in the fractions
corresponding to the 60S ribosomal
particle but not in the polysome frac-
tions (Fig. 2A) suggesting that Jjj1, like
Rei1, is a pre-60S-associated factor. Since Jjj1 and Rei1
interact in a two-hybrid assay and since they are both
associated to pre-60S ribosomal particles, we attempted to
determine if both proteins were present in the same
particles. We purified the complex associated with Jjj1-
TAP using a strain producing a Rei1–13myc fusion protein
and also performed the reverse experiment. Jjj1–13myc was
only present in the complexes associated with Rei1 but not
with Rlp24 (Fig. 2B) and Rei1–13myc was copurified with
Jjj1-associated comp lexes but not with Rlp24. As a control,
we observed that Rlp24 was copurified neither with Jjj1-
TAP nor with Rei1-TAP while it copurified with Mak11-
TAP. These results suggest that (1) Jjj1 and Rei1 are present
in the same complexes and (2) like Rei1, Jjj1 binds to the
particle after the release of Rlp24.
We checked for the dependence of the presence of Jjj1 for
the binding of Rei1 to the particle and inversely by using
strains depleted for the first one and pro ducing a TAP
fusion of the second one. The association of the proteins to
the pre-60S complexes was tested by Western blott ing on
fractions of a sucrose gradient. Surprisingly, neither Jjj1 nor
Rei1 was required for the recruitment of the other protein
to the pre-60S particle (data not shown).
Jjj1 and Rei1 are functionally linked
Several lines of evidence indicate that there is a strong
similarity between Jjj1 and Rei1: (1) Both proteins are
FIGURE 2. JjjI and Rei1 are involved in the same complexes. (A) Jjj1 is associated with the
60S particles. The whole-cell extract of a strain expressing a Jjj1-TAP fusion protein (OT52)
was separated by ultracentrifugation on a 10%–50% sucrose gradient. Fractions of 0.5 mL were
collected. Proteins were precipitated by TCA and separated on a SDS–4%–12% polyacrylamide
gel. The presence of Jjj1-TAP fusion protein was revealed by Western blotting using PAP
antibodies (Sigma). (B) Jjj1 and Rei1 are present in the same pre-60S complexes. TAP puri-
fications were performed on strains expressing Rei1-TAP, Jjj1-13myc (ED46); Jjj1-TAP, Rei1–
13myc (ED19); Rlp24-TAP, Jjj1–13myc (ED87); and Rlp24-TAP, Rei1–13myc (LMA352)
fusion proteins. Mak11-TAP (LMA375) and Jjj1–13myc (ED82) strains were used as control.
The presence of Jjj1–13myc, Rei1–13myc, Rlp24, and Rpl1 was tested using antibodies against
myc, Rlp24, and Rpl1, respectively.
Jjj1 is required for pre-60S factors recycling
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1573
Page 4
involved in the pre-60S biogenesis, (2)
they are located in the cytoplasm, (3)
they physically interact, (4) both pro-
teins have Zn finger motifs, and (5)
deletion of either JJJ1 or REI1 leads to a
cold sensitive phenotype. To determine
if these proteins would have redundant
functions, we tested the effect of the
double deletion (Fig. 3A). In compari-
son to a generation time of 2 h 41 min
for a wild-type strain, we observe a
generation time of 5 h 20 min for jjj1D
rei1D double mutant strain (ED21)
similar to the generation time of 5 h
37 min of the rei1D strain (LMA523),
whereas the generation time for jjj1D is
of 4 h 5 min, suggesting that both JJJ1
and REI1 genes are epistatic.
To further investiga te the role of Jjj1,
we performed a high-copy suppressor
genetic screen with the JJJ1-deleted
strain (ED10) at 20°C. Interestingly, in
addition to JJJ1 containing plasmids,
one family of the rescued plasmids
containing the REI1 gene was able to
partially complements the cold sensitiv-
ity of the jjj1D mutant strain. Figure 3B
(left panel) shows the complementation
using a 2 mm plasmid derived from
pFL44L containing JJJ1. As expected,
overexpression of REI1 was also able
to partially restore the col d-sensitive
phenotype of jjj1D (Fig. 3B, left panel).
We tested the reverse situation by over-
expressing JJJ1 into a rei1D mutant
strain (ED72). While pFL44L-REI1
complements the absence of Rei1, the
overexpression of JJJ1 had no effect on
the rei1D mutant (same as empty vec-
tor; data not shown). Overexpression of
either JJJ1 or REI1 was tested on the
jjj1D rei1D double mutant strain (ED21) (Fig. 3B, right
panel). While the overexpression of JJJ1 had no effect,
we observed a partial rescue of the growth of jjj1D rei1D
double mutant strain by overexpression of REI1.
Polysome profiles of the jjj1D strains transformed with
different vectors were analyzed after a shift of temperature
for 2 h at 23°C from 30
°C. The amount of 60S subunit was
clearly affec ted in the absence of Jjj1 as compared to the
jjj1D strain complemented by the overexpression of JJJ1.
Interestingly, REI1 overexpression partially restored the
relative amount of 60S subunit (Fig. 3C), in agreement
with the partial growth rescue.
We conclude that overexpression of REI 1 partially
complements the absence of Jjj1 but the reverse is not true.
Therefore, while Jjj1 and Rei1 proteins display some
similarities and are involved in the same pathway, their
functions are not identical.
The absence of either Arx1 or Alb1 is able to complement
the growth defect of the rei1D strain (Lebreton et al. 2006b;
Meyer et al. 2007). To further investigate the similarities
and the differences between Jjj1 and Rei1, we checked
whether the presence of Arx1 or Alb1 was responsible fo r
the cold sensitivity of the jjj1D strain. In fact the jjj1D arx1D
and jjj1D alb1D double mutant strains grew as well as
arx1D or alb1D strains at 23°C (Fig. 4A), showing
that the absence of Arx1 (or Alb1) rescued the cold
sensitivity of jjj1D. Moreover, using polysome profiles, we
observed that the relative amount of 60S was restored to the
FIGURE 3. Rei1 and Jjj1 are genetically linked. (A) Deletion of both JJJ1 and REI1 genes is
epistatic. Ten times serial dilutions of wild-type (BY4741), rei1D (LMA523), jjj1D (ED10), and
rei1D jjj1D (ED21) culture were spotted on solid rich medium. Their growth on YPGlu at 23°C
was compared. Generation times are indicated. (B) REI1 overexpression partially complements
the jjj1D growth defect. A jjj1D (ED10) strain (left panel) and a rei1D jjj1D (ED21) strain (right
panel) were transformed with either the empty vector pFL44L or pFL44L-REI1 or pFL44L-JJJ1.
Growth differences were illustrated by 10 times serial dilutions on solid minimal medium
without uracil at 23°C for 2 d. Generation times are indicated. (C) REI1 overexpression
partially restores 60S amount of jjj1D strain. jjj1D strain (ED10) was transformed with either
pFL44L (left panel) or pFL44L-REI1 (middle panel) or pFL44L-JJJ1 (right panel). The strains
were grown in minimal media without uracil at 30°C and shifted to 23°C for 2 h. The whole-
cell extracts were separated by ultracentrifugation on a 10%–50% sucrose gradient. Absorbance
at 254 nm was measured. Peaks corresponding to the various subunits and polysomes
are indicated.
Demoinet et al.
1574 RNA, Vol. 13, No. 9
Page 5
wild-type level in the jjj1D arx1D and jjj1D alb1D double
mutant strains when compared to jjj1D alone (Fig. 4B).
This observation is consistent with the recent hypothesis
that the accumulation of Arx1 and Alb1 as a small
cytoplasmic complex (Lebreton et al. 2006b) could be
responsible for the slow growth phenotype of the rei1D
strain.
Jjj1 and Rei1 have distinct functions
in the Arx1/Alb1 recycling
Since the cold sensitivity of the two deleted jjj1D or rei1D
strains was abolished by the deletion of ARX1 or ALB1,we
hypothesized that Jjj1 could participate with Rei1 in the
recycling of shuttling pre-60S factors. We predicted that the
proteins affected in their recycling in the absence of Rei1
would also be affected by the absence of Jjj1. We observed
that Arx1-GFP and Alb1-GFP accumu-
lated in the cytoplasm in the absence of
Jjj1 as well as in the absence of Rei1
(Fig. 5A, left and middle panels). To
check if Arx1 and Alb1 are blocked on
the cytoplasmic pre-60S particles or
present in small cytoplasmic complexes
in the absence of Jjj1, whole-cell extracts
from strains expressing Alb1-GFP in the
absence of Jjj1 were separated by sedi-
mentation on a sucrose gradient. In the
absence of Jjj1, endogenous Arx1 and
Alb1-GFP fusion protei n sedimented in
the 60S peak whereas in the absence of
Rei1, both Arx1 and Alb1-GFP accu-
mulated as small complexes (Fig. 5B) as
previously described (Lebreton et al.
2006b). Similar results were obtained
in a strain expressing an Arx1-GFP
fusion protein (data not shown). These
results are in agreement with the genetic
links described above. Even if Jjj1 and
Rei1 are involved in the same pathway,
their functions are not fully equivalent.
Previous results have suggested that the
recycling of these factors involved the
Kap121 karyopherin pathway. This
hypothesis was supported by the fact
that overexpression of KAP121 was able
to recycle Arx1 and Alb1 into the
nucleus and partially restore the slow
growth phenotype of the rei1D strain
(Fig. 6A, left panel; Lebreton et al.
2006b). If in jjj1D, Arx1 and Alb1 are
not released from the particle, over-
expression of KAP121 should not result
in relocalization of Arx1/Alb1 in the
nucleus. Indeed, in contrast to rei1D,
KAP121 overexpression (Fig. 6A, middle panel) had no
effect on the jjj1D strain. It was able to restore neither the
growth phenotype nor the polysome profile (data not
shown) nor the normal localization of Arx1-GFP (Fig.
6B). As a control, we observed that, in a rei1D strain,
KAP121 overexpression allowed Arx1/Alb1 recycling (Fig.
6B). Interestingly, KAP121 overexpression led to a partial
rescue of the slow growth phenotype in the jjj1D rei1D
double mutant strain as in rei1D strain (Fig. 6A, cf. right
and left panels).
These results confirm that in the absence of Jjj1, Arx1
and Alb1 are mostly blocke d on pre-60S cytoplasmic
particles, where they are probably not available to the
import machinery, whereas in the absence of Rei1, Arx1
and Alb1 accumu late as small cytoplasmic complexes,
where they still can be caught by an overwhelming amount
of Kap121 karyopherin.
FIGURE 4. In the absence of Arx1/Alb1, deletion of JJJ1 is not toxic. (A) Deletion of either
ARX1 or ALB1 rescues the cold sensitivity effect of jjj1D strain. Ten times serial dilutions of
wild-type (BY4741), jjj1D (ED10), arx1D (LMA539), alb1D (LMA525), arx1D jjj1D (ED52),
and alb1D jjj1D (ED65) cultures were spotted on solid rich medium. Their growth on YPGlu
at 23 °C was compared. (B) Deletion of either ARX1 or ALB1 restores a wild-type polysome
profile. The whole-cell extracts from the strains used in A were separated on a sucrose gradient
as described in Figure 1B.
Jjj1 is required for pre-60S factors recycling
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1575
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The absence of Jjj1 or Rei1 differently affects
Tif6 recycling
We recently proposed that, in a rei1D strain, the cytoplas-
mic accumulation of small Arx1-associated complexes
could be responsible for blocking Tif6 on the pre-60S
particle (Lebreton et al. 2006b). In the absence of Jjj1, no
such small cytop lasmic Arx1-associated complexes were
formed; we therefore expected Tif6 to be released from the
pre-60S particle and correctly recycled. Interestingly, while
Tif6 accumulated in the cytoplasm in the absence of Rei1, it
was localized in the nucleus in the absence of Jjj1 (Fig. 5A,
right panel). We also checked the sedimentation of Tif6-
TAP in the absence of Jjj1; it sedimented in fractions
corresponding to the 60S peak (Fig. 5B). This suggests that
in the absence of Jjj1, Tif6 is correctly released from the
pre-60S cytoplasmic particles and recycles to the nucleus.
In conclusion, Tif6 does not seem to be affected by the
deletion of JJJ1 while it requires REI1 for dissociation from
the cytoplasmic pre-60S particle.
DISCUSSION
The first precursor particle involved in ribosome biogen-
esis, the 90S, generates the pre-40S and pre-60S interme-
diate ribosomal particles after the A2 cleavage. The large
ribosomal subunit maturation requires about 80 pre-ribo-
somal factors. This pre-60S particle goes through successive
maturation steps, including rRNA processing, conforma-
tional changes, and transport events through the nucleolus,
the nucleoplasm, and the nucleopore complex. Thus, it is
not surprising that most of these 80 or so pre-60S factors are
nuclear factors whereas few are strictly cytoplasmic. When
the pre-60S particles reach the cytoplasm, the mature ribo-
somal RNAs are formed, and many pre-ribosomal factors
have already left the particles. Only a few shuttling factors,
such as Arx1, Alb1, Tif6, Rlp24, and Nmd3, are still present.
Then, some strictly cytoplasmic pre-60S factors act on the
pre-60S particles to finalize their maturation.
We report here the role of a strictly cytoplasmic pre-60S
factor, Jjj1, in the biogenesis of the large ribosomal subunit.
FIGURE 5. In the absence of Jjj1, Arx1/Alb1 are blocked on cytoplasmic 60S particles. (A) Arx1 and Alb1 but not Tif6 (B) accumulate into the
cytoplasm in the absence of Jjj1. Arx1-GFP (LMA401), Alb1-GFP (LMA545-B), Arx1-GFP, jjj1D (ED49), Arx1-GFP, rei1D (LMA411), Alb1-GFP,
jjj1D (ED66), Alb1-GFP, rei1D (LMA545-A), Tif6-TAP (ED72), Tif6-TAP, rei1D (ED92), and Tif6-TAP, jjj1D (ED89) strains were grown in
minimal media at 30°C and shifted to 23°C for 2 h. The cells were observed by fluorescence microscopy. Tif6-TAP localization was observed on
fixed cells using PAP antibodies (Sigma) at 1/5000 dilution followed by Cy3-conjugated secondary antibodies (Jackson Immunoresearch) at 1/250
dilution. (C) Arx1 and Alb1 are blocked on the 60S particle in the absence of Jjj1. The whole-cell extracts from the strains used in A were separated
on a sucrose gradient and the fractions were analyzed as described in Figure 2A. The Western blots were performed using the fractions of the
polysome profiles from 2 to 15. The presence Arx1, Tif6, and Alb1-GFP fusion proteins were checked using anti-Arx1, anti-Tif6, and anti-GFP
antibodies (Santa Cruz Biotechnology) at 1/700, 1/5000 and 1/700 dilutions, respectively.
Demoinet et al.
1576 RNA, Vol. 13, No. 9
Page 7
While this manuscript was being prepared, Meyer et al.
(2007) reported a functional analysis of Jjj1. In agreement
with our data, Meyer et al. report that Jjj1 is involved in the
biogenesis of the 60S large ribosomal subunit and shares
similar features with the Rei1 pre-60S factor. Additional
experiments presented here revealed that, while the func-
tions of Jjj1 and Rei1 proteins show similarities, they are
nevertheless clearly distinct.
While Rei1 and Jjj1 share similar features, they have
distinct functions
In addition to the fact that Jjj1 and Rei1 have zinc-finger
motifs, both proteins share several similarities. They are
late strictly cytoplasmic pre-60S associated facto rs. Their
absence leads to a cold sensitivity and a slow growth
phenotype correlated with a relative decrease of the amount
of 60S subunit and a weak defect in rRNA maturation
(Figs. 1, 3; Hung and Johnson 2006; Lebreton et al. 2006b;
Meyer et al. 2007). Both proteins interact together not only
in a two-hybrid assay (Fig. 1) but also in co-immunopre-
cipitation experiments (Fig. 2), reveal-
ing that both proteins are biochemically
associated to similar pre-60S complexes.
Since both Jjj1 and Rei1 are physically
associated to the same cytoplasmic late
particles, we tried to define the order of
assembly of these two factors to the pre-
60S particles. Surprisingly, sucrose gra-
dient analysis of strains delete d for REI1
or JJJ1 revealed that the binding of each
one of these proteins to the pre-60S
particles is independent from the pres-
ence of the other (data not shown).
Here, we observed that, as for the
recruitment of Rei1 onto the particle,
the binding of Jjj1 is one of the la st steps
in the ribosome biogenesis. Jjj1, like
Rei1, binds to the particle after the
release of Rlp24 pre-60S factor from
the particles. Thus, the transient pres-
ence of Jjj1 on pre-60S particles corre-
lates very well with the chaperone
activity of Jjj1.
Besides the biochemical features of
Jjj1 and Rei1, several data indicate that
the roles of these proteins are inter-
twined. Both Jjj1 and Rei1 are involved
in the recycling of the pre-60S shuttling
factors Arx1 and Alb1 (Fig. 5A). The
slow growth phenotype of jjj1D or rei1D
strains can be rescued by the deletion
of ARX1 or ALB1 (Fig. 4; Hung and
Johnson 2006; Lebreton et al. 2006b),
suggesting that the fate of Arx1 and
Alb1 is responsible for the cold sensitivity of jjj1D and rei1D
strains. As reported by Meyer et al. (2007), we observe that
Arx1 and Alb1 also accumulate in the cytoplasm in the
absence of Jjj1.
However, while Jjj1 and Rei1 proteins have strong
similarities, we describe here key differences revealing
distinct func tions of these proteins in 60S formation. Our
previous model proposed that, in the absence of Rei1, it
is the cytoplasmic accumulation of the small complexes
including Arx1 and Alb1 that prevents the release of Tif6
from the pre-60S particle. We show here that, in the
absence of Jjj1, Arx1 and Alb1 do not accumu late in small
cytoplasmic complexes but remain associated with the pre-
60S particle and that Tif6 is correctly recycled to the
nucleus (Fig. 5).
Three karyopherins, Kap121/Pse1, Kap123, and Kap104
(Rout et al. 1997; Leslie et al. 2004) are involved in the
import of ribosomal components into the nucleus. In a
rei1D strain, overexpression of KAP121 partially restores
the growth defect and allows the recycling of Arx1/Alb1 and
Tif6 into the nucleus (Lebreton et al. 2006b) while in the
FIGURE 6. KAP121 overexpression has distinct effects on rei1D and jjj1D strains. (A) KAP121
overexpression does not restore the growth of jjj1D. Ten times serial dilutions of reiD
(LMA523), jjj1D (ED10), and rei1D jjj1D (ED21) transformed with either the empty vector
pFL44L or pFL44L-REI1 or pFL44L-JJJ1 or pFL44L-KAP121 were spotted on solid minimal
medium without uracil at 23°C for 2 d. Generations times are indicated. (B) KAP121
overexpression does not restore nuclear localization in the absence of Jjj1. Arx1-GFP, jjj1D
(ED49) and Arx1-GFP, rei1D (LMA411) strains were transformed with either the empty vector
pFL44L or pFL44L-REI1 or pFL44L-JJJ1 or pFL44L-KAP121. The cells were grown in minimal
media at 30°C and shifted at 23°C for 2 h. Arx1-GFP localization was observed by fluorescence
microscopy.
Jjj1 is required for pre-60S factors recycling
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1577
Page 8
absence of Jjj1, overexpression of KAP121 has no effect on
Arx1 and Alb1 recycling (Fig. 6). This is correlated with the
fact that Arx1 and Alb1 are not released from pre-60S
particles in this context.
We conclude that Jjj1 would be preferentially involved in
the dissociation of Arx1 and Alb1 from the pre-60S particle,
whereas Rei1 would rather be preferentially involved in the
recycling of these factors by the karyopherins pathway.
Why is the presence of Arx1 toxic when JJJ1 or REI1
are deleted?
Arx1 deficient cells have almost no growth phenotype.
Surprisingly, the absence of Arx1 leads to halfmers forma-
tion, whereas the amount of 60S does not seem strongly
affected (Fig. 4) suggesting that this factor is not directly
required for the production of the pre-60S subunit but
could rather be important for the quality of the produced
60S subunit. The data presented here and by Hung and
Johnson (2006), Lebreton et al. (2006b), and Meyer et al.
(2007) suggest that a failure in its recycling may be more
deleterious to the cells than its absence. Indeed, we have
described two mutants (rei1D and jjj1D) whose deleterious
phenotypes are correlated with the cytoplasmic accumula-
tion of Arx1 and Alb1 and are rescued by deletion of the
ARX1 or ALB1 gene. Therefore, it appears that the
cytoplasmic accumulation of Arx1 is mainly responsible
for the growth defects observed in the absence of either Jjj1
or Rei1. But we now show that the deleterious effect occurs
both when Arx1 and Alb1 are released from the pre-60S
(rei1D) or stalled on the pre-60S particle (jjj1D). The
recycling of Tif6 is also affected in the absence of Rei1 as
Arx1 and Alb1 accumulate in small cytoplas mic complexes.
Surprisingly, nuclear Arx1 recycling is not necessarily
correlated with a rescue of a normal growth of the cells.
Indeed, in the absence of Rei1, JJJ1 (data not shown) and
KAP121 overexpressions allow the recycling of Arx1 into
the nucleus (Lebreton et al. 2006b). However, only KAP121
overexpression is able to partially rescue the wild-type
phenotype. In all cases, Arx1 is not anymore associated to
the pre-60S particle and the polysome profile is not
rescued. When the correct Arx1 process is interrupted,
whatever the gene overexpressions tested to rescue the
wild-type phenotype, only partial function of Arx1 is
recovered, suggesting that the moment and the conditions
in which Arx1 and Alb1 are released from the particles and
reimported are crucial. Jjj1, as a Hsp40 chaperone catalyz-
ing structural transitions, could play a key role in allowing
the comm itment of the ribosomal large subunit toward
translation initiation.
The role of Jjj1 as a chaperone
The role of Hsp40, characterized by their J domain, is to
stimulate the ATPase activity of their Hsp70 partner.
Recently, Meyer et al. (2007) have shown that the Hsp70
cochaperone of Jjj1 could be Ssa1. In eukaryotic cells, there
are two major cytosolic classes of Hsp70: the SSA family
composed of fourgenes, SSA1–4, and the SSB family,
encoded by SSB1 and SSB2. Whereas Ssb chaperones are
ribosome associated and bind nascent polypeptides to
prevent their misfolding at the ribosome exit channel, Ssa
chaperones have pleiotropic functions: translocation of the
newly synthetized polypeptides to the reticulum endoplas-
mic or mitochondria and other folding process.
Apart from Jjj1 , no chaperones have been found to be
specifically involved in the biogenesis of the ribosomal large
subunit. Indeed, the RAC (ribosome associated complex),
composed of the Hsp40, Zuo 1, and its two Hsp70 partners,
Ssz1 and Ssb1/2, binds the NAC (nascent chain associated
complex), composed of three factors, Egd2, Egd1 and Btt1,
to prevent misfolding of the newly synthesized polypeptide
at the exit ribosome channel; it is not involved in the
maturation of the ribosomal subunits. The specif ic role of
Jjj1 in ribosome biogenesis could be compared to the
specific function of the auxilin Hsp40. Indeed, Swa2/auxilin
Hsp40 is specifically involved in clathrin uncoating
by inducing transconformational changes of clathrin
(Ungewickell et al. 1995; Fan et al. 2003; Fotin et al.
2004; Hennessy et al. 2005). Jjj1 seems to be specifically
involved in Arx1 release from the particle. Indeed, when the
very conserved histidine–proline–aspartic acid (HPD)
motif of the J domain is mutated, Arx1 cannot be recycled
and accumulates in the cytoplasm (Meyer et al. 2007). This
suggests that the function of Jjj1 relies on its chaperone
activity.
CONCLUSION
When the pre-60S particles arrive in the cytoplasm, they
are no t yet competent for translation and a few final steps
are required. A strong cytoplasmic control is important
because commitment of pre-60S particles into translation
initiation should be extremely deleterious for the cells. A
coordinated pre-60S particles export and pre-60S factors
import should be an important level of regulation to
control the entry in translation.
MATERIALS AND METHODS
Plasmids, oligonucleotides, strains,
and growth conditions
The strains used in this study are listed in Table 1. Chromosomal
insertions were obtained by homologous recombination using
PCR fragments in MGD13-353D or BY strains (Baudin et al.
1993). Standard yeast genetic methods and selective growth media
were used. The plasmids encoded JJJ1 as two-hybrid bait (pED1)
or prey (pED4) were obtained by Gateway cloning JJJ1 in pAS2DD
and pACTIIst destination vectors. The other two-hybrid plasmids
Demoinet et al.
1578 RNA, Vol. 13, No. 9
Page 9
were indicated by Lebreton et al. (2006a,b).
We constructed an empty vector pED13,
which was derived from pFL44L plasmid,
by elimination of an insert of the library
by Kpn1/Sal1 digestion. The pED12 plasmid
was generated by cloning JJJ1 extending
from 350 base pairs upstream from the
initiation codon to 640 bp downstream of
the stop codon at the Not1 site. Generation
times were calculated from growth curves in
liquid culture at 23°C during a period of 32 h.
Sucrose gradient sedimentation
and Western blotting
The strains were grown at 30°C in appro-
priate medium to an A
600
of 0.3–0.6, and the
mutant strains were shifted at 23°C for 2 h
before cycloheximide treatment. Extracts
were prepared in 10 mM Tris-HCl (pH
7.4), 30 mM MgCl
2
, 100 mM NaCl, and
50 mg/mL cycloheximide using glass beads
vortexing. They were fractioned by ultracen-
trifugation in 10%–50% sucrose gradients
for 3 h at 39,000 rpm at 4°C in an SW41 Ti
rotor. The fractions were recovered with an
ISCO fractionator, and the absorbance at
254 nm was measured. The proteins were
precipitated with 10% TCA and separated
on polyacrylamide-SDS gels.
Nog1 and Rlp24 (Saveanu et al. 2003),
Nog2 (Saveanu et al. 2001), Tif6 (Senger
et al. 2001), Rpl1 (a gift of F. Lacroute, Gif
sur Yvette, France), and Nmd3 (a gift of A.W.
Johnson, Austin, TX, USA) native proteins
were detected by indirect immunoblotting,
using specific polyclonal rabbit antibodies
at a 1:5000 dilution (except at 1:10,000 for
Rpl1). Specific polyclonal rabbit antibodies
against Arx1 and Rei1 were used at 1:700
and 1:2000, respectively (Lebreton et al. 2006b).
TAP-tagged proteins were revealed with a
1:10,000 dilution of peroxidase-antiperoxi-
dase complex (PAP; Sigma). GFP-tagged
and 13Myc-tagged proteins were detected
using anti-GFP 1:700 (rabbit polyclonal
antibody, Santa Cruz) and anti-myc 1:1000
(mouse monoclonal antibody, Santa Cruz),
respectively. Secondary antibodies (Goat Anti-
Rabbit- or Goat Anti-Mouse-HRP Conjugate
from Bio-Rad) were used at a 1:10,000 dilu-
tion. Peroxidase activity of the secondary anti-
bodies was revealed using Millipore chemilu-
minescence HRP substrate system.
High copy number suppressor
genetic screen
The jjj1D strain (ED11) was transformed
with a yeast genomic library cloned into a
TABLE 1. Yeast strains used in this study
Strain Genotype Reference
MGD353-13D MATa, trp1-289, ura3-52, ade2,
leu2,-3-112, arg4
Rigaut et al. (1999)
BY4741 MATa, ura3D0, his3D1, leu2D0, met15D0 Brachmann et al. (1998)
BY4742 MATa, ura3D0, his3D1, leu2D0, lys2D0 Brachmann et al. (1998)
ED10 MATa, ura3D0, his3D1, leu2D0,
met15D0, jjj1D
T
KanMX4
Winzeler et al. (1999)
ED11 MATa, ura3D0, his3D1, leu2D0, lys2D0,
jjj1D
T
KanMX4
Winzeler et al. (1999)
ED70 MATa, ura3D0, his3D1, leu2D0,
met15D0, rei1D
T
KanMX4
Winzeler et al. (1999)
ED71 MAT a, ura3D0, his3D1, leu2D0, lys2D0,
rei1D
T
KanMX4
Winzeler et al. (1999)
ED21 MATa, ura3D0, his3D1, leu2D0, lys2D0,
met15?, rei1D
T
NatMX4,
jjj1D
T
KanMX4
This study
ED52 MATa, ura3D0, his3D1, leu2D0,
met15D0, arx1D
T
Hygro,
jjj1D
T
KanMX4
This study
ED65 MATa, ura3D0, his3D1, leu2D0,
met15D0, alb1D
T
NATMX4,
jjj1D
T
KanMX4
This study
LMA523 MATa, ura3D0, his3D1, leu2D0,
met15D0, rei1D
T
NATMX4
Lebreton et al. (2006b)
LMA539 MATa, ura3D0, his3D1, leu2D0,
met15D0, arx1D
T
Hygro
Lebreton et al. (2006b)
LMA525 MATa, ura3D0, his3D1, leu2D0, lys2D0,
alb1D
T
NATMX4
Lebreton et al. (2006b)
LMA401 MATa, ura3D0, his3D1, leu2D0,
met15D0, ARX1–GFP/His3MX6
Lebreton et al. (2006b)
LMA411 MATa, ura3D0, his3D1, leu2D0,
met15D0, ARX1–GFP/His3MX6,
rei1D
T
KanMX4
Lebreton et al. (2006b)
ED49 MATa, ura3D0, his3D1, leu2D0,
met15D0, ARX1–GFP/His3MX6,
jjj1D
T
KanMX4
This study
LMA545-B MATa, ura3D0, his3D1, leu2D0, lys2D0,
met15D0, ALB1-GFP/His3MX6
Lebreton et al. (2006b)
LMA545-A MATa, ura3D0, his3D1, leu2D0, lys2?,
met15?, ALB1-GFP/His3MX6,
rei1D
T
KanMX4
Lebreton et al. (2006b)
ED66 MATa, ura3D0, his3D1, leu2D0, lys2?,
met15?, ALB1-GFP/His3MX6,
jjj1D
T
KanMX4
This study
OT52 MATa, ura3D0, his3D1, leu2D0,
met15D0, JJJ1-TAP/His3MX6
Winzeler et al. (1999)
ED19 MATa, trp1-289, ura3-52, ade2,
leu2,-3-112, arg4, JJJ1-TAP/klTRP1,
REI1-13Myc/KanMX6
This study
ED46 MATa, trp1-289, ura3-52, ade2,
leu2,-3-112, arg4, REI1-TAP/URA3,
JJJ1-13Myc/klTRP1
This study
ED87 MATa, trp1-289, ura3-52, ade2,
leu2,-3-112, arg4, RLP24-TAP/ klTRP1,
JJJ1-13Myc/KanMX6
This study
LMA352 MATa, trp1-289, ura3-52, ade2,
leu2,-3-112, arg4, RLP24-TAP/klTRP1,
REI1-13Myc/KanMX6
Lebreton et al. (2006b)
ED82 MATa, trp1-289, ura3-52, ade2,
leu2,-3-112, arg4, JJJ1-13Myc/KanMX6
This study
(continued)
Jjj1 is required for pre-60S factors recycling
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1579
Page 10
(URA3) pFL44L plasmid. The transformants were isolated on
minimum medium plate without uracil (URA). The plates were
incubated at 30°C overnight and then shifted at 20°C for several
days. The clones that display a slow growth rescue at 20°C were
selected by comparing the size of the transformants with those
transformed with an empty vector. Their plasmidic DNAs were
extracted and DNA inserts were sequenced using M13 forward
and reverse universal primers. The suppressor plasmids were
tested by retransformation of the jjj1D strain (ED11).
Fluorescence microscopy
Cells were grown in minimal medium overnight at 30°Ctoan
A
600
of 0.5. The mutant strains were shifted to 23° C for 2 h.
Indirect immunofluorescence of the TAP-tagged fusion proteins
was detected using anti-protein A antibodies and Cy3 secondary
antibodies as described (Pringle et al. 1991). Fluorescence was
visualized using an epifluorescence microscope (model DMRB;
Leica) as described by Lebreton et al. (2006b).
RNA extraction, Northern blotting,
and primer extension
After growth of each yeast strain in rich media at 30°Cuptoan
A
600
of 0.5, the cultures were shifted at 23°C for 15 min or 2 h.
The cultures were centrifuged, and yeast cells were broken using
glass beads. RNA extractions were performed with phenol/chlo-
roform. Mature and intermediate large species were separated on
1% agarose gel and small species were separated on 5% poly-
acrylamide–urea gels. RNAs were transferred on Hybond+
membranes, and their identification was performed by hybridiza-
tion with various
32
P-labeled oligonucleotides. Primer extensions
were performed using
32
P-labeled oligonucleotides, and the prod-
ucts were separated on 5% polyacrylamide–urea gels. The se-
quences of the oligonucleotides were previously described by
Saveanu et al. (2001).
Two-hybrid matrix
The strain CG1945 transformed with the bait Gateway plasmid
pAS2DD-JJJ1, REI1, ARX1, ALB1, and RPL24B was mated with the
strain Y187 transformed with the prey Gateway plasmid pActII
containing the same open reading frame (ORF). Diploids were
selected on minimal medium without leu-
cine and tryptophan (LW) plates, and the
diploids displaying a positive two-hybrid
interaction were selected on minimal
medium without leucine, tryptophan, and
histine (LWH) plates. An X-Gal overlay
was performed according to Fromont-
Racine et al. (1997) to select the positive
two-hybrid clones on the second reporter
gene, the LacZ gene.
Tandem affinity purification
Purifications were performed from 4 L of
yeast culture as described by Rigaut et al.
(1999) using classical buffers containing
0.1 M NaCl. The TEV eluates were precipitated
with 10% TCA, and the final TAP purifica-
tions were precipitated with methanol/chloroform. The purified
complexes were separated on a 4%–12% polyacrylamide gradient–
SDS gel and analyzed by Western blotting as described above.
ACKNOWLEDGMENTS
We thank Jean-Christophe Rain (Hybrigenics, Paris, France) for
performing the yeast two-hybrid screen with Rei1 as bait, Alice
Lebreton (CGM, Gif-sur-Yvette, France) for strains and plasmids,
and Caty Gonzalez for technical help. We are grateful to Francxois
Lacroute (CGM, Gif-sur-Yvette, France) for providing the high-
copy vector genomic S. cerevisiae library, Edouard Bertrand (IGM,
Montpellier, France) for providing the two-hybrid Gateway
plasmids, and Franco Fasiolo (IBMC, Strasbourg, France), Arlen
W. Johnson (MGM, Austin, TX, USA), and Francxois Lacroute for
the gift of Tif6, Nmd3, and Rpl1 antibodies, respectively. We
thank Helen Neil for critical reading of the manuscript and
Cosmin Saveanu for all the scientific discussions and advice for
experiments. E.D. and G.L. are grateful to Claire Bonnerot for
having introduced them to Jjj1. This work was supported by a
grant from the Ministe
`
re de
´
le
´
gue
´
a
`
l’Enseignement Supe
´
rieur et a
`
la Recherche (ACI-BCM0089-2003) to M. F.-R., ARC (33.49) and
FRM (FRM-INE20031114111) to G.L. E. D. received a fellowship
from La ligue Nationale contre le Cancer.
Received March 27, 2007; accepted June 12, 2007.
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TABLE 1. Continued
Strain Genotype Reference
LMA375 MATa, ura3D0; his3D1; leu2D0; met15D0;
lys2D0, Mak11-TAP/His3MX6
Saveanu et al. (2007)
ED72 MATa, ura3D0, his3D1, leu2D0,
met15D0, TIF6-TAP/His3MX6
Winzeler et al. (1999)
ED89 MATa, ura3D0, his3D1, leu2D0,
met15D0, TIF6-TAP/His3MX6,
jjj1D
T
KanMX4
This study
ED92 MATa, ura3D0, his3D1, leu2D0,
met15D0, TIF6-TAP/His3MX6,
rei1D
T
KanMX4
This study
Question marks indicate that the genotype was not determined for this auxotrophy marker
in this strain.
Demoinet et al.
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Jjj1 is required for pre-60S factors recycling
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    • "Mutations in Rei1 and Jjj1 lead to accumulation of Arx1 on pre-60S subunits in the cytosol (Hung and Johnson, 2006; Lo et al., 2010; Meyer et al., 2007, 2010) and block downstream pre-60S maturation events, including the release of the anti-association factor Tif6p (the yeast homolog of human eIF6) from the interface side of the 60S subunit (Lebreton et al., 2006; Lo et al., 2010). These defects can be alleviated by deletion of Arx1 (Hung and Johnson, 2006; Lebreton et al., 2006; Lo et al., 2010) or Alb1 (Demoinet et al., 2007; Lebreton et al., 2006; Meyer et al., 2007), a small, highly positively charged protein that forms a stable complex with Arx1 and is thereby recruited to the maturing pre-60S particle in the nucleoplasm (Bradatsch et al., 2007, 2012; Lebreton et al., 2006). These observations indicate that events at the tunnel exit are coordinated with maturation steps at the subunit interface and that the presence of Arx1 and Alb1 is inhibitory to downstream maturation. "
    [Show abstract] [Hide abstract] ABSTRACT: Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.
    No preview · Article · Dec 2015 · Cell
  • Source
    • "The nacΔjjj1Δ cells lacking Jjj1 and all three genes encoding NAC showed no synthetic growth phenotype compared to jjj1Δ cells under the conditions tested (Fig 5B). Ribosome profiles of jjj1Δ and nacΔjjj1Δ cells (Fig 5C–5F ) revealed that the deletion of JJJ1 resulted in a decrease of 60S subunits and in the appearance of halfmers (Fig 5E) , indicating that this strain has a ribosome biogenesis defect as described previously [27, 28]. Loss of NAC in jjj1Δ cells did neither enhance the halfmer formation nor cause a further reduction of 60S, 80S or polysome peaks. "
    [Show abstract] [Hide abstract] ABSTRACT: Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β'-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ'-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ'-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ'-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β'-NAC or α-NAC subunits as well as αβ'-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.
    Full-text · Article · Nov 2015 · PLoS ONE
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    • "Upon reaching the cytoplasm, pre-ribosomal subunits undergo final maturation steps that include the release of export factors and, similar to nucleoplasmic biogenesis events, energy-driven enzymes play key roles in these processes [187,188]. For example, dissociation of the CRM1 adaptor Nmd3 requires the GTPase Lsg1, while the AAA-ATPase Drg1 works together with the ATPase Ssa1 to bring about the release and recycling of Arx1189190191192. How the recruitment, action and release of the numerous export adaptors implicated in pre-ribosomal subunit export are coordinated is not yet fully understood . "
    [Show abstract] [Hide abstract] ABSTRACT: RNAs and ribonucleoprotein complexes (RNPs) play key roles in mediating and regulating gene expression. In eukaryotes, most RNAs are transcribed, processed and assembled with proteins in the nucleus and then either function in the cytoplasm or also undergo a cytoplasmic phase in their biogenesis. This compartmentalisation ensures that sequential steps in gene expression and RNP production are performed in the correct order and allows important quality control mechanisms that prevent the involvement of aberrant RNAs/RNPs in these cellular pathways. The selective exchange of RNAs/RNPs between the nucleus and cytoplasm is enabled by nuclear pore complexes (NPCs), which function as gateways between these compartments. RNA/RNP transport is facilitated by a range of nuclear transport receptors and adaptors, which are specifically recruited to their cargos and mediate interactions with nucleoporins to allow directional translocation through NPCs. While some transport factors are only responsible for the export/import of a certain class of RNA/RNP, others are multifunctional and, in the case of large RNPs, several export factors appear to work together to bring about export. Recent structural studies have revealed aspects of the mechanisms employed by transport receptors to enable specific cargo recognition, and genome-wide approaches have provided the first insights into the diverse composition of pre-mRNPs during export. Furthermore, the regulation of RNA/RNP export is emerging as an important means to modulate gene expression in stress conditions and disease.
    Full-text · Article · Oct 2015 · Journal of Molecular Biology
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