The specialized cytosolic J-protein, Jjj1, functions in 60S ribosomal subunit biogenesis.
ABSTRACT J-proteins and Hsp70 chaperones function together in diverse cellular processes. We identified a cytosolic J-protein, Jjj1, of Saccharomyces cerevisiae that is associated with 60S ribosomal particles. Unlike Zuo1, a 60S subunit-associated J-protein that is a component of the chaperone machinery that binds nascent polypeptide chains upon their exit from the ribosome, Jjj1 plays a role in ribosome biogenesis. Cells lacking Jjj1 have phenotypes very similar to those lacking Rei1, a ribosome biogenesis factor associated with pre-60S ribosomal particles in the cytosol. Jjj1 stimulated the ATPase activity of the general cytosolic Hsp70 Ssa, but not Ssb, Zuo1's ribosome-associated Hsp70 partner. Overexpression of Jjj1, which is normally approximately 40-fold less abundant than Zuo1, can partially rescue the phenotypes of cells lacking Zuo1 as well as cells lacking Ssb. Together, these results are consistent with the idea that Jjj1 normally functions with Ssa in a late, cytosolic step of the biogenesis of 60S ribosomal subunits. In addition, because of its ability to bind 60S subunits, we hypothesize that Jjj1, when overexpressed, is able to partially substitute for the Zuo1:Ssb chaperone machinery by recruiting Ssa to the ribosome, facilitating its interaction with nascent polypeptide chains.
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ABSTRACT: The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. In this review, we summarize our current understanding of nuclear export of pre-ribosomal subunits and cytoplasmic maturation steps that render pre-ribosomal subunits translation-competent.Chromosoma 05/2014; · 3.26 Impact Factor
The specialized cytosolic J-protein, Jjj1, functions
in 60S ribosomal subunit biogenesis
Alison E. Meyer*, Nai-Jung Hung†, Peizhen Yang*, Arlen W. Johnson†, and Elizabeth A. Craig*‡
*Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, WI 53706; and†Section of Molecular Genetics and Microbiology
and Institute of Molecular Biology, University of Texas, Austin, TX 78712
Contributed by Elizabeth A. Craig, December 3, 2006 (sent for review November 14, 2006)
J-proteins and Hsp70 chaperones function together in diverse
cellular processes. We identified a cytosolic J-protein, Jjj1, of
Saccharomyces cerevisiae that is associated with 60S ribosomal
particles. Unlike Zuo1, a 60S subunit-associated J-protein that is a
component of the chaperone machinery that binds nascent
polypeptide chains upon their exit from the ribosome, Jjj1 plays a
similar to those lacking Rei1, a ribosome biogenesis factor associ-
ated with pre-60S ribosomal particles in the cytosol. Jjj1 stimulated
the ATPase activity of the general cytosolic Hsp70 Ssa, but not Ssb,
Zuo1’s ribosome-associated Hsp70 partner. Overexpression of Jjj1,
which is normally ?40-fold less abundant than Zuo1, can partially
rescue the phenotypes of cells lacking Zuo1 as well as cells lacking
Ssb. Together, these results are consistent with the idea that Jjj1
normally functions with Ssa in a late, cytosolic step of the biogen-
esis of 60S ribosomal subunits. In addition, because of its ability to
bind 60S subunits, we hypothesize that Jjj1, when overexpressed,
is able to partially substitute for the Zuo1:Ssb chaperone machin-
ery by recruiting Ssa to the ribosome, facilitating its interaction
with nascent polypeptide chains.
Hsp70 ? Rei1 ? ribosome biogenesis ? Hsp40
ones participate in a wide variety of cellular functions, including
protein folding, translocation of proteins across organellar mem-
branes, and disassembly of protein complexes (1). An Hsp70’s
interaction with client proteins is modulated by its nucleotide-
binding state, with ATP fostering rapid interaction with client
proteins. This interaction is then stabilized upon ATP hydrolysis
(2). J-proteins play a critical role by stimulating the ATPase
activity of their Hsp70 partner through interaction of their
J-domains, thereby promoting association of the Hsp70 with the
The number of J-proteins in a particular cellular compartment
typically exceeds that of Hsp70s (1). The cytosol of the yeast
Saccharomyces cerevisiae contains 13 J-proteins (3) but only two
classes of Hsp70s: the Ssas (encoded by SSA1–4) and Ssbs
(encoded by SSB1–2) (4). The Ssa class has a variety of cellular
functions, including translocation of proteins into the endoplas-
mic reticulum and mitochondria, uncoating of clathrin-coated
vesicles, and general protein folding (4). A number of different
J-proteins have been shown to work with Ssa in these processes.
On the other hand, the more specialized Ssbs, which are pre-
dominantly ribosome-associated, can be cross-linked to very
short nascent polypeptides (5) and are thought to function in the
protection of newly synthesized polypeptides as they exit the
ribosome (6, 7). Unlike the Ssas, which are essential, cells lacking
the Ssbs grow slowly, particularly at low temperatures, and are
hypersensitive to cations (8).
Although all J-proteins contain the canonical J-domain, some
have additional sequences that allow them to carry out special-
ized functions. In some cases these sequences may serve to tether
the J-protein to a particular cellular location or to recruit
additional factors that are important for their function. For
-proteins, also known as Hsp40s, are obligate cochaperones of
Hsp70-type molecular chaperones. Together, these chaper-
example, Zuo1 is tethered to the ribosome, where it functions as
the Ssbs’ J-protein partner (9). Strains lacking the Ssbs, Zuo1, or
Ssz1, a regulatory protein that forms a heterodimer with Zuo1,
have the same phenotypes, consistent with the three proteins
acting together as a chaperone machine (5, 6).
During our analysis of the J-proteins of S. cerevisiae, we found
that an uncharacterized J-protein of the yeast cytosol, Jjj1,
contains a region, in addition to the J-domain, that is similar to
a region of Zuo1. Therefore, we sought to determine Jjj1’s
function. We found that Jjj1, which is ?40-fold less abundant
than Zuo1, is also ribosome-associated. However, Jjj1 has a role
independent of Zuo1 in the biogenesis of 60S ribosomal sub-
units. Despite this unique role of Jjj1, overexpression of Jjj1 can
partially rescue the growth defects of cells lacking Zuo1, indi-
cating a partial overlap in function between these two J-proteins.
Jjj1 and Zuo1 Comigrate with 60S Ribosome Subunits. A comparison
of the amino acid sequence of the ribosome-associated J-protein
Zuo1 with other J-proteins of S. cerevisiae revealed an 82-aa
region between amino acids 205 and 287 that shares 50%
similarity with amino acids 179–261 of Jjj1. Other than the
J-domains, the remainder of Zuo1 and Jjj1 are strikingly differ-
ent. Unlike most J-proteins, Zuo1 has an internal J-domain.
in positively charged amino acids. On the other hand Jjj1, like
most J-proteins, has an N-terminal J-domain. In addition, Jjj1
has two putative C2H2-type zinc fingers that flank a region rich
in charged amino acids, although, in the case of Jjj1, this region
has an overall negative charge.
The 82-aa region that is conserved between Jjj1 and Zuo1 is
not present in other J-proteins of S. cerevisiae. This unique
similarity motivated us to ask whether Jjj1, like Zuo1, is a
was prepared in magnesium-free buffer from WT cells and
centrifuged through a magnesium-free sucrose gradient to sep-
arate the 40S and 60S ribosomal subunits (10). The fractions
were subjected to immunoblot analysis. Jjj1 and Zuo1 comi-
grated almost exclusively with 60S subunits, consistent with 60S
association (Fig. 1A). Furthermore, Rpl8, a protein of the 60S
subunit, could be coimmunoprecipitated with Jjj1, confirming
that Jjj1 associates with ribosomes (Fig. 1B)
apparent ribosome association of both Zuo1 and Jjj1 raised the
question as to whether Zuo1 and Jjj1 perform overlapping
functions. To test this idea, we first placed the coding region of
Jjj1 under the control of the constitutive GPD promoter, which
led to an ?60-fold overexpression of Jjj1 (data not shown), or an
performed research; A.E.M. contributed new reagents/analytic tools; A.E.M., P.Y., A.W.J.,
and E.A.C. analyzed data; and A.E.M., A.W.J., and E.A.C. wrote the paper.
The authors declare no conflict of interest.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2007 by The National Academy of Sciences of the USA
January 30, 2007 ?
vol. 104 ?
estimated 130,000 molecules per cell (11). ?zuo1 cells grow
slowly, particularly at 30°C and below, and are hypersensitive to
a wide variety of cations, including Na?, Li?, and the cationic
translation-inhibiting drug, paromomycin, compared with WT
suggesting that Jjj1 can, in part, substitute for Zuo1 (Fig. 1C).
function for Jjj1, was also true, we first created a ?jjj1 strain and
tested for phenotypic effects of the absence of Jjj1. Deletion of
JJJ1 also resulted in cold sensitivity (Fig. 2A). This function of
Jjj1 depends on its cochaperone activity, because a mutant
protein in which the highly conserved histidine–proline–aspartic
acid (HPD) motif of the J-domain was replaced with three
expressed at levels similar to WT protein (Fig. 2B, data not
shown). The ?jjj1 strain did not show any hypersensitivity to
NaCl or paromomycin (Fig. 2A). We then tested whether
overexpression of Zuo1 affected the cold sensitivity of ?jjj1 cells.
However, neither increased expression of Zuo1 alone nor Zuo1
and Ssz1 together obviously improved the growth of ?jjj1 cells,
suggesting that Jjj1 carries out a cellular function distinct from
that of Zuo1 (Fig. 2C).
?jjj1 Cells Have a Defect in 60S Ribosomal Subunit Biogenesis. The
possibility that Jjj1 performs a function different from Zuo1 led
us to look more carefully at the issue of ribosome association.
Total cellular extract from WT cells was separated on a sucrose
gradient, and fractions were analyzed for the presence of Jjj1,
previous results (12), Zuo1 comigrated with ribosomes and was
found distributed throughout the gradient in fractions contain-
ing free 60S subunits, 80S monosomes and polysomes, similar to
perhaps, 80S particles, with some trailing into the polysomal
fractions (Fig. 3A). This difference in fractionation also pointed
to a difference in function between Jjj1 and Zuo1.
Comigration of Jjj1 with 60S subunits and the slow growth at
low temperatures of cells lacking Jjj1 is reminiscent of Rei1, a
recently identified cytosolic factor required for biogenesis of 60S
subunits (13–15). Deletion of REI1 causes a decrease in 60S
levels and the appearance of ‘‘half-mer’’ ribosomes (14, 15).
Half-mers are interpreted as 48S preinitiation complexes stalled
on mRNA because of a lack of mature large ribosomal subunits,
which can result from either decreased levels of mature 60S
subunits or from a 40S–60S subunit joining defect (16). Based on
these similarities, we decided to analyze cellular extracts from a
?jjj1 strain and, as a control, a ?rei1 strain, grown at 23°C, by
sucrose gradient centrifugation. Compared with a WT strain, the
?jjj1 and ?rei1 mutants showed a significant accumulation of
half-mer ribosomes (Fig. 3B). In contrast, analysis of extracts
from a ?zuo1 strain grown at 23°C did not, consistent with the
inability of increased expression of Zuo1 to rescue the cold
sensitivity of ?jjj1.
Strains having defects in 60S subunit biogenesis often have
reduced steady-state levels of mature 60S. To assess the levels of
60S subunits in the ?jjj1 strain, total cellular extracts from WT,
?jjj1, and ?rei1 cells grown at 23°C were prepared and analyzed
in magnesium-free buffer to separate 40S and 60S subunits. The
area under each peak was measured, and the 60S/40S ratio was
determined for each strain. The 60S/40S ratio for the WT strain
was 1.92 ? 0.06, similar to the previously reported ratio of 2 (17).
For ?rei1, the 60S/40S ratio was 1.36 ? 0.09 and for ?jjj1, 1.44 ?
did WT cells. The ?rei1 strain, which has been reported to have
fewer 60S subunits (14, 15), had a similar reduction in 60S
rescues ?zuo1. (A) Lysate from a WT strain was prepared in magnesium-free
(Upper). Fractions were collected and analyzed for the presence of Jjj1, Zuo1,
and Rpl3 by immunoblotting (Lower). (B) Extracts from cells containing plas-
mid borne Jjj1-HA or, as a control, genomically integrated Arx1-HA, were
incubated with anti-HA antibody and protein A beads. Precipitated proteins
were eluted from protein A beads in sample buffer and separated by SDS/
as immunoprecipitation from an untagged strain (C) were included as con-
trols. Immunoblotting was performed by using antibodies specific for the HA
or Rpl8 epitopes. (C) Tenfold serial dilutions of cells were spotted on minimal
media plates with indicated additions and incubated at indicated tempera-
tures; pRS415GPD JJJ1 (1JJJ1); paromomycin (Paro).
Overexpression of Jjj1, which comigrates with 60S subunits, partially
equal numbers of cells of the indicated genotypes were diluted 10-fold, spotted
on plates, and incubated under conditions indicated on rich media (A), minimal
media; pRS416TEF ZUO1 (1ZUO1); pRS415GPD SSZ1 (1SSZ1) (C).
Jjj1 is a J-protein with a function distinct from Zuo1. Approximately
Meyer et al.
January 30, 2007 ?
vol. 104 ?
no. 5 ?
subunits (30%). The 60S/40S ratio for the ?zuo1 strain was
indistinguishable from that of WT, 2.03 ? 0.08.
Absence of the 60S Biogenesis Factor Arx1 Suppresses the ?jjj1
Phenotype. Although factors involved in the biogenesis of the
large subunit often cosediment with free 60S subunits, some act
in the nucleus and are exported with the pre-60S particle to the
cytosol. These shuttling factors are then reimported into the
nucleus after their release from the 60S particle. Jjj1 has been
reported to be cytosolic (18). To determine whether Jjj1 is
potentially a shuttling pre-60S factor, we used a leptomycin B
(LMB)-sensitive strain harboring the T539C mutation in the 60S
exporter Crm1. If Jjj1 shuttles, we would expect it to be trapped
in the nucleus when ribosome export is blocked by addition of
LMB. However, in the presence of the Crm1 inhibitor LMB, Jjj1
remained cystosolic (Fig. 3C), whereas the 60S export adapter
Nmd3 relocalized to the nucleus, as reported (19, 20). We
conclude that Jjj1 functions solely in the cytosol.
Because Rei1 is also a solely cytosolic factor involved in 60S
ribosome biogenesis, and both ?rei1 and ?jjj1 cells have a
cold-sensitive phenotype, we compared the growth of the indi-
vidual mutants with that of the double mutant, ?jjj1 ?rei1. ?rei1
cells grew slightly less well at 23°C compared with ?jjj1 cells.
However, ?jjj1 ?rei1 cells grew indistinguishably from cells
lacking only Rei1 (Fig. 4A), suggesting to us that Jjj1 and Rei1
might function together, because loss of a second protein in a
pathway often does not cause additional harm to the cell.
Rei1 has been reported to play a role in the cytosol-to-nuclear
recycling of pre-60S biogenesis factors, such as Arx1, that are
bound to pre-60S particles upon their exit to the cytosol (14, 15).
In a WT strain, the majority of the fluorescent signal produced
by Arx1-GFP is found in the nucleus, with a faint cytoplasmic
signal being consistent with nuclear-cytoplasmic transport of
Arx1-bound pre-60S subunits (14, 15). In the absence of Rei1,
however, Arx1-GFP is primarily cytosolic (14, 15), and the
protein persists on the 60S ribosomal subunit (14), suggesting
that it is not recycled properly to the nucleus.
To determine whether Arx1 is recycled properly in a ?jjj1 strain,
we monitored the Arx1-GFP signal in this strain. Similar to ?rei1,
at 25°C Arx1-GFP is predominantly found in the cytosol in ?jjj1
cells, suggesting that it may remain bound to the 60S subunit (Fig.
4B). Indeed, in the absence of Jjj1, Arx1 still comigrated with 60S
ribosomal subunits (Fig. 4C), and the 60S ribosomal protein Rpl8
was coimmunoprecipitated with Arx1 (Fig. 4D), indicative of
role of Jjj1 depends on its cochaperone activity, because Arx1-GFP
ribosomes at 23°C. (A) Ten OD254units of lysate from WT cells grown at 30°C
(Upper). Fractions collected were analyzed by immunoblotting for the pres-
ence of Jjj1, Zuo1, and Rpl3 (Lower). (B) Ten OD254units of lysate from the
indicated strains grown at 23°C were centrifuged through 7–47% sucrose
gradients, and the OD254was monitored. (C) Log-phase cells of the LMB-
sensitive strain AJY1539 were incubated in LMB (0.1 ?g/ml) for 30 min and
then fixed with formaldehyde and subjected to indirect immunofluorescence
monitored by using anti-rabbit rhodamine-conjugated antibodies. DAPI was
used to visualize nuclei.
Cells lacking Jjj1, a strictly cytosolic protein, accumulate half-mer
and incubated at 23°C. (B) Localization of Arx1-GFP was monitored by fluo-
rescence microscopy in the indicated strains. All cells were grown at 25°C in
rich media, except ?jjj1 carrying pRS316 jjj1HPD3AAA(jjj1HPD3AAA), which was
cultured in minimal media lacking uracil. (C) Lysates from 25°C-grown cells
containing chromosomally integrated ARX1-HA were centrifuged through
7–47% sucrose gradients and the OD254monitored (Upper). Fractions were
analyzed for the presence of Arx1-HA and Rpl8 by immunoblot analysis
(Lower). (D) Extracts from WT or ?jjj1 cells containing genomically integrated
Arx1-HA were incubated with anti-HA antibody and protein A beads. Precip-
itated proteins were eluted from protein A beads in sample buffer and
separated by SDS/PAGE. Total protein extract (T) as well as immunoprecipita-
tion from an untagged strain (C) were included as controls. Immunoblotting
was performed by using antibodies specific for the HA or Rpl8.
www.pnas.org?cgi?doi?10.1073?pnas.0610704104 Meyer et al.
is also primarily cytosolic in the jjj1HPD3AAAmutant (Fig. 4B). The
fact that, in the absence of Jjj1, Arx1 migrates further into the
participate, at least marginally, in subunit joining and thus trans-
lation initiation. Regardless, we conclude that Jjj1 is required for
proper recycling of Arx1.
The deletion of ARX1 has also been shown to suppress the
growth defect of a ?rei1 strain (14, 15), presumably because the
continued presence of Arx1 on the 60S subunit is detrimental to
the final stages of 60S maturation (14). Therefore, we compared
the phenotypes of ?jjj1, ?arx1, and ?jjj1 ?arx1 strains. ?arx1
cells, although slightly cold sensitive, grew better at 23°C than
?jjj1 cells. ?jjj1 ?arx1 cells grew as well as ?arx1 cells (Fig. 5A).
Thus deletion of ARX1 suppresses the cold-sensitive growth
defect caused by the absence of either Rei1 or Jjj1. The absence
of Arx1 has also been reported to substantially alleviate the
half-mer accumulation of a ?rei1 strain (14). We tested the effect
of deletion of ARX1 on the polysome profile of a ?jjj1 cells.
Distinct half-mer peaks were absent in profiles of ?jjj1 ?arx1
cells. (Figs. 3B and 5B). In addition, the ratio of 60S to 40S
subunits in ?jjj1 ?arx1 was 1.76 ? 0.07 compared with 1.92 ?
0.06 in WT and 1.44 ? 0.05 in ?jjj1. We conclude that deletion
of ARX1 at least partially relieved the 60S biogenesis defect of
a ?jjj1 strain. The fact that the phenotype of ?jjj1 is more severe
than deletion of ARX1 suggests that the consequences of per-
sistent association of Arx1 with 60S subunits may be more severe
than those for its complete absence.
Arx1 is reported to exist in a stable complex with the small,
highly basic protein Alb1, and deletion of ALB1 also suppresses
the growth defects of a ?rei1 strain (15). We tested the effects
of the absence of Alb1 in our system. Deletion of ALB1
suppressed the half-mer defect (Figs. 3B and 5B), the cold-
sensitivity of a ?jjj1 strain (Fig. 5A), and the decrease in 60S
subunits, as the 60S/40S ratio of ?jjj1 ?alb1 cells was 1.71 ? 0.04,
consistent with the idea that Arx1 and Alb1 function together.
on the growth of a ?zuo1 strain (data not shown).
Jjj1 J-Domain Stimulates the ATPase Activity of Ssa, but Not Ssb. The
proper localization of Arx1 depends on the J-domain of Jjj1.
Because no J-protein has been found to function alone, but
rather always with an Hsp70 partner, we sought to determine the
Hsp70 partner of Jjj1. Because Jjj1 and Zuo1 have similarities in
their amino acid sequences, and Zuo1 functions with the ribo-
some-associated Hsp70 Ssb, we first asked whether strains
lacking Ssb have a defect in 60S subunit biogenesis, which would
be expected if Jjj1 and Ssb functioned together. Total cellular
extracts from a ?ssb1/2 strain grown at 23°C were analyzed. The
polysome profile of this strain was similar to that of a WT strain,
with no visible accumulation of half-mer ribosomes (Fig. 6A).
This lack of evidence for a role of the Ssb-type Hsp70s in
ribosome biogenesis suggested to us that Jjj1 did not function
with Ssb. We then asked whether overexpression of Jjj1 could
partially rescue ?zuo1 even in the absence of Ssb1/2. Not only
could expression of Jjj1 from the GPD promoter rescue a ?zuo1
?ssz1 strain, which lacks the entire ribosome-associated chap-
erone complex (Fig. 6B, data not shown).
The results reported above suggested to us that Jjj1 and Ssb
Hsp70 of the cytosol. We could not test cells lacking Ssa, as we
did those lacking Ssb, because such strains are nonviable. In
addition, reduced Ssa function results in pleiotropic effects,
including defects in the nucleolus (21), the major site of ribo-
some biogenesis. However, a critical role of a J-protein is the
stimulation of the ATPase activity of its partner Hsp70 and thus,
such stimulation serves as an indicator of functional partnerships
between J-proteins and Hsp70s. Therefore, we purified Jjj1 and
tested whether it was able to stimulate the ATPase activity of
Ssa1 or Ssb1, using preformed, radiolabeled ATP-bound Hsp70
complexes. Even at a 0.1:1 ratio with Ssa1, Jjj1 stimulated Ssa1’s
ATPase activity 5-fold, with 17-fold stimulation occurring at a
0.5:1 ratio (Fig. 6C). This level of stimulation was equivalent to
that obtained with Ydj1, a known cytosolic J-protein partner of
Ssa1 (9, 22). This stimulation depended on a functional J-
did not significantly stimulate the ATPase activity of Ssb1,
expected (9). These results suggest that Ssa is the in vivo Hsp70
partner of Jjj1.
The results reported here strongly support the idea that the
cytosolic J-protein Jjj1 plays an important role in a late cytosolic
Approximately equal numbers of cells of the indicated genotypes were di-
OD254units of lysate from the indicated strains grown at 23°C were centri-
fuged through 5–50% sucrose gradients and the OD254was monitored.
Deletion of ARX1 or ALB1 suppresses defects of ?jjj1 cells. (A)
from the ?ssb1 ? ssb2 (?ssb1/2) grown at 23°C were centrifuged through a
5–50% sucrose gradient, and the OD254was monitored. (B) Approximately
equal numbers of cells of the indicated genotypes were diluted 10-fold and
complexes were incubated in the presence of various concentrations of Jjj1,
Jjj1HPD3AAA,Ydj1, or Zuo1–Ssz1 heterodimer. The rates of hydrolysis of ATP
were determined, and fold stimulation over the basal rate was plotted.
Meyer et al.
January 30, 2007 ?
vol. 104 ?
no. 5 ?
step in the biogenesis of 60S ribosomal subunits. First, Jjj1 comi-
grates with 60S subunits. Second, ?jjj1 cells are cold sensitive and
have a decrease in the amount of 60S subunits compared with WT
cells. Third, analysis of ?jjj1 polysome profiles revealed the pres-
ence of half-mers. These phenotypes are strikingly similar to those
of cells lacking Rei1, a cytosolic factor required in a late step of 60S
ribosome biogenesis. It has been postulated that the cytosolic
protein Rei1 facilitates removal of two proteins that are also
nucleus bound to the pre-60S subunit, allowing subsequent steps of
subunit maturation (14, 15). In good part, this hypothesis is based
on the fact that in the absence of Rei1, Arx1 remains ribosome-
associated (14), and both Arx1 and Alb1 fail to recycle to the
nucleus (14, 15). Additionally, deletion of either ARX1 or ALB1
suppresses the defects of a ?rei1 mutant (14, 15). Intriguingly, Arx1
also remains ribosome-associated, and fails to recycle, in ?jjj1 cells.
The growth defect of ?jjj1 is suppressed by the absence of Arx1 or
Alb1 as well.
Given that ?jjj1 and ?rei1 cells have nearly identical pheno-
types, how might Jjj1 be functioning in ribosome biogenesis? It
is possible that Rei1 and Jjj1 function together, perhaps directly
or indirectly causing a conformational change that reduces the
affinity of Arx1/Alb1 for the pre-60S ribosome, causing their
dissociation. There are precedents for J-protein:Hsp70 machin-
ery being used for the remodeling of protein–nucleic acid and
protein–protein complexes. For example, DnaJ and DnaK of
Escherichia coli are required for dissociation of ?P protein from
initiation complex to allow phage ?DNA replication (23); in
eukaryotic cells, the J-protein auxilin and Hsc70 are required for
uncoating of clathrin-coated vesicles (24). The dissociation/
recycling of two other pre-60S ribosomal biogenesis factors,
Nmd3 and Tif6, from the cytosol are thought to require the
GTPases, Lsg1 and Efl1, respectively (25–27). In addition,
ATPases of the AAA-ATPase and ABC-family ATPases are
required in 60S biogenesis (28, 29). Thus, it is possible that in all
of these cases, energy from the hydrolysis of nucleotides may be
used for remodeling of pre-60S ribosomal particles to facilitate
generation of mature subunits.
The association of two J-proteins, Zuo1 and Jjj1, with the
ribosome raises questions of overlap in function as well as
whether they share an Hsp70 partner. It is well established that
Zuo1 functions in partnership with Ssb (9). On the other hand,
our data point to Jjj1 partnering with Ssa. This conclusion is
supported by the ability of Jjj1 to stimulate the ATPase activity
of Ssa but not Ssb. We have no evidence that Zuo1 plays a role
in ribosome biogenesis, because ?zuo1 cells do not accumulate
half-mers. In addition, increased expression of Zuo1 does not
suppress the defects observed in cells lacking Jjj1. On the other
hand, increased expression of Jjj1 partially suppressed both the
slow growth and the cation-sensitive phenotype of cells lacking
Zuo1. We found that overexpression of Jjj1 was not only able to
rescue the ?zuo1 strain, but ?ssb1/2, ?ssb1/2 ?zuo1, and ?ssb1/2
and Jjj1 function with different Hsp70 partners.
The data reported here, together with previously published
data, suggest to us the following scenario. Normally, Jjj1 pref-
erentially associates with cytosolic pre-60S ribosomal particles
and, with Ssa as its Hsp70 partner, facilitates ribosome biogen-
esis, perhaps aiding in the recycling of factors such as Alb1 and
Arx1. Such a function is consistent with its predominant comi-
gration with 60S subunits. Zuo1, which is ?40 times more
abundant than Jjj1 and present in a 1:1 stoichiometry with
ribosomes, binds near the ribosome exit site, and, working with
its regulatory factor Ssz1 and its Hsp70 partner Ssb, facilitates
early stages of protein folding. Although perhaps not a normal
occurrence, Jjj1 may also be competent to bind to mature 60S
subunits. Indeed, when Jjj1 is overexpressed 20-fold, it all
remains ribosome associated (data not shown). Jjj1 may be able
to recruit the Hsp70 Ssa to ribosomes, fostering its interaction
with nascent polypeptide chains and allowing this chaperone
machinery to substitute for Zuo1/Ssb in protein folding. Such an
ability of Ssa to perform the function of Ssb is reminiscent of the
ability of the human Zuo1 ortholog, Mpp11, to substitute for
Zuo1 (30). In this case, Mpp11 partners with the highly con-
served Ssa, rather than the fungal-specific Ssb. Mpp11, which
stimulates the ATPase activity of Ssa, but not Ssb, can rescue
both a ?zuo1 and a ?zuo1 ?ssb1/2 mutant (30).
Our unpublished results suggest that Jjj1 and Zuo1 binding to
the ribosome may not be mutually exclusive. Even so, the ability
of Jjj1 to substitute for Zuo1 may not be surprising, because
precise positioning of Hsp70 at the exit site may not be required
reported that the peptidyl-prolyl isomerase Trigger Factor, a
ribosome-associated chaperone on the E. coli ribosome, can
functionally substitute for the Zuo1/Ssb/Ssz1 machinery, even
though they appear to bind to different sites, because their
binding to the ribosome is not mutually exclusive (21). However,
Trigger Factor does bind close to the ribosome exit site, through
its interaction with Rpl23, the ortholog to Rpl25 of eukaryotes
(31). Evidence suggests that Rei1 may associate with pre-60S
subunits in the vicinity of the exit tunnel, as Rpl25-GFP, but not
Rpl25 tagged with the smaller HA epitope, blocks association of
Arx1 with the ribosome (14). Because Jjj1 also is important for
proper recycling of Arx1 and can perform some of the functions
of Zuo1, which is thought to function with Ssb in nascent chain
protection, it is tempting to speculate that it may also be located
near the exit of the ribosome tunnel.
Jjj1’s function in ribosome biogenesis appears to be specific.
However, despite the ability of Jjj1 to partially substitute for
Zuo1, the basis of such specificity, be it in its positioning on the
ribosome or its direct interaction with other factors as well as the
exact mechanism of action of J-protein:Hsp70 machinery in
ribosome biogenesis awaits further study.
Materials and Methods
Yeast Strains, Plasmids, and Genetic Techniques. JJJ1 was obtained
using Pfu Turbo polymerase (Stratagene, La Jolla, CA) and
cloned into the XbaI and BamHI sites in pRS415GPD (32) to
generate pRS415GPD JJJ1. WT and mutants of JJJ1, generated
by using QuikChange (Stratagene), were cloned into pRS316
(33). To create Jjj1-HA, 3 tandem copies encoding the hemag-
JJJ1. For Zuo1 overexpression, DNA from positions 1–1802 was
amplified and cloned into pRS415TEF by using ClaI and
BamHI, to generate pRS415TEF ZUO1. To overexpress SSZ1,
codons 1–2617 were PCR amplified and cloned into the BamHI
and SalI sites of pRS416GPD to generate pRS416GPD SSZ1.
To obtain a null allele of JJJ1, positions ?215 to 2115 of the
jjj1::KanMX4 cassette were PCR amplified from a homozygous
diploid yeast genomic knockout collection (Open Biosystems,
Huntsville, AL) (34) and transformed into DS10 (GAL2 his3-
11,15 leu2-3112 lys1 lys2 ?trp1 ura3-52), a derivative of S288C.
The KanMX4 cassette was replaced with TRP1 through a marker
swap plasmid (35). ?rei1, ?arx1, and ?alb1 were obtained by
PCR amplification of the KanMX4-marked genes from the
create multiple deletion strains, such as ?ssb1::HIS3
?ssb2::TRP1 ?zuo1::URA3 and ?ssb1::HIS3 ?ssb2::TRP1
?zuo1::URA3 ?ssz1::LYS2, appropriate haploid strains were
mated and sporulated (36). Strains having ARX1 tagged with
three tandem copies of the HA epitope or GFP inserted in the
chromosome at the ARX1 locus were made by homologous
recombination in WT BY4741 and ?jjj1 DS10 (37).
Minimal and rich glucose-based media were as described (8).
Plates without additions, or with addition of paromomycin (100
www.pnas.org?cgi?doi?10.1073?pnas.0610704104 Meyer et al.
?g/ml), were incubated for 2 days at 30°C or 3 days at 23°C,
unless otherwise indicated. Plates having 0.5 M NaCl were
incubated for 3 days at 30°C. In cases where strains were to be
plated on minimal selective media, cells were first transformed
with the appropriate empty vector for comparison with strains
having test plasmids. All chemicals were obtained from Sigma
(St. Louis, MO) unless otherwise stated.
Analysis of Cell Extracts. Separation of 40S and 60S subunits under
low-magnesium conditions was performed as described (10),
with slight modifications. One liter of the appropriate yeast
culture was grown in yeast peptone dextrose (YPD) medium at
23°C to an optical density of 0.5–1 at 600 nm. Cells were
harvested by centrifugation at 4°C and washed with 14 ml of
ice-cold breaking buffer B [50 mM Tris?HCl (pH 7.4)/50 mM
NaCl/1 mM dithiolthreitol]. Cells were centrifuged at 4,000 ? g
for 5 min and resuspended in 3.5 ml of breaking buffer B plus 140
units of rNAsin (Promega, Madison, WI). Lysis of cells and
clearing of lysate was performed as described (12). Ten OD254
units of cleared lysate were layered on 10-ml 15–30% sucrose
gradients prepared in breaking buffer B. Gradients were cen-
trifuged in an SW40 Ti rotor at 4°C at 40,000 rpm for 4.5 h.
Fractions of 0.6 ml were collected, and proteins were precipi-
tated with 86% acetone overnight at ?20°C before immunoblot
analysis. For analysis of 60S/40S ratios, profile images were
scanned at identical resolutions, and the areas under each peak
were quantified by using NIH Image v1.63. Lysate preparation
and sucrose gradient centrifugation for polysome analysis was
carried out essentially as described (12, 26).
Jjj1 Purification. WT and mutant JJJ1 were PCR amplified such
that DNA encoding a His tag was inserted at the C terminus.
DNA was digested with NdeI and BamHI and then ligated into
the pET3a vector (Novagen, San Diego, CA). The plasmids were
transformed into BL21 pLys cells lacking DnaK and DnaJ for
protein expression. Proteins were induced at 30°C with the
addition of 1 mM IPTG (isopropyl-?-D-thiogalactopyranoside)
at an OD600of 0.6 and incubated for an additional 4 h. The cell
lysates were prepared by French press, and purification was
performed following the His-tag protein purification protocols
Other Methods. Other procedures were carried out as described:
isolation of Hsp70–ATP complexes and ATPase assays (9),
microscopy (14), indirect immunofluorescence experiments
(19), and LMB experiments (26). Immunoblot detection was
carried out by using the ECL system (Amersham Pharmacia
Biotech, Piscataway, NJ) according to the manufacturer’s sug-
gestion. The anti-Jjj1 antibody was raised in rabbits against a
fusion of amino acid 304–590 of Jjj1 to GST. Anti-Rpl3 was a gift
from Jon Warner (Albert Einstein College of Medicine, Bronx,
NY). Anti-HA was purchased from Covance (Denver, PA).
Anti-Rpl8, anti-Nmd3, and anti-Zuo1 were produced as re-
ported (12, 19, 38).
We thank J. Warner for antibodies specific for L3. This work was
supported by National Institutes of Health Grants GM31107 (to E.A.C.)
and GM53655 (to A.W.J.).
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