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Vol. 14, Issue 8, 3266-3279, August 2003
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* Research Center for Materials Science, Nagoya University, Nagoya, 464-8602,
Japan;
Department of Chemistry, Graduate School of Science, Nagoya University,
Nagoya, 464-8602, Japan
Submitted November 22, 2002;
Revised April 4, 2003;
Accepted April 4, 2003
Monitoring Editor: Thomas Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Trotta and
Abelson, 1999; Wolin and
Matera, 1999; Hopper and
Phizicky, 2003). The splicing of tRNA introns is carried out by
sequential action of three enzymes: tRNA splicing endonuclease
(Peebles et al.,
1979; Rauhut et al.,
1990), tRNA ligase (Phizicky
et al., 1986), and 2'-phosphotransferase
(Culver et al., 1997).
The yeast tRNA endonuclease that catalyzes the first step of tRNA splicing
consists of four subunits, Sen54p, Sen2p, Sen34p, and Sen15p. Sen2p and Sen34p
have catalytic centers that cleave a 5'-exon-intron junction and an
intron-3'-exon junction, respectively
(Trotta et al.,
1997). Both of the subunits show significant homology to
homooligomeric archaeal tRNA endonuclease
(Li et al., 1998). In
contrast to its soluble counterparts in archaea and Xenopus
(DeRobertis et al.,
1981; Li et al.,
1998), the yeast enzyme has been regarded as an integral membrane
protein, because the enzymatic activity is associated with membranes and its
extraction requires detergents (Peebles
et al., 1983). Sen2p, which has a hydrophobic stretch
suitable to traverse membranes, was thought to anchor the other subunits to
the inner nuclear envelope (NE; Trotta
et al., 1997). tRNA ligase was also shown to be localized
on the inner periphery of the NE (Clark and
Abelson, 1987).
tRNA splicing has been believed to occur just before the export of mature
tRNAs. The following observations suggested a "coupling model," in
which the spliced tRNAs are directly handed to the nuclear export machinery
(Peebles et al.,
1983; Sharma et al.,
1996). In yeast, mutants defective in mature tRNA export, like
rna1 and los1, concomitantly accumulate end-matured,
unspliced pre-tRNAs in the nucleus (Hopper et al.,
1978,
1980). Rna1p is a Ran GAP
homologue in yeast (Corbett et
al., 1995), and Los1p, a homologue of exportin-t, is an
export carrier of tRNAs (Arts et
al., 1998; Hellmuth
et al., 1998; Kutay
et al., 1998). Defects in some nucleoporins also result
in pre-tRNA accumulation (Sharma et
al., 1996). The nuclear localization of the accumulated
pre-tRNAs in these mutants was demonstrated by fluorescence in situ
hybridization (FISH; Sarkar and Hopper,
1998; Sarkar et al.,
1999; Grosshans et
al., 2000).
However, this coupling model has been challenged by several observations.
In Xenopus oocytes, aminoacylation of functional tRNAs by
aminoacyl-tRNA synthetase (RS) appears to take place in the nucleus before
their export (Lund and Dahlberg,
1998). A similar system operates in yeast. In
nup116 mutant cells, the mature tRNAs that accumulated in the
nucleus were already aminoacylated (Sarkar
et al., 1999). Conversely, the blockade of a certain RS
by a mutation or a specific inhibitor caused the accumulation of the
corresponding mature tRNA, but not the pre-tRNA, in the nucleus
(Grosshans et al.,
2000). A mutation within the nuclear localization signal (NLS) of
TyrRS also caused the nuclear accumulation of tRNA-Tyr, without affecting its
aminoacylation ability (Azad et
al., 2001). These results are against the direct coupling
between the splicing and the export of tRNAs. Besides, under certain
conditions, los1 cells accumulate only pre-tRNAs, not mature tRNAs,
in the nucleus (Feng and Hopper,
2002). Without tight coupling between the splicing and the export,
we need to seek another mechanism to explain accumulation of unspliced
pre-tRNAs in the export mutants.
In this situation, it is essential to know the exact place and timing in which the tRNA splicing occurs. Therefore, we decided to reexamine the localization of the tRNA splicing endonuclease in yeast. Unexpectedly, the endonuclease was present mainly on the mitochondrial surface, and several lines of evidence indicate that the mitochondrial pool of the enzyme has positive roles in tRNA splicing. On the basis of these results, we propose a new possibility that pre-tRNAs are spliced on the mitochondrial surface after their export out of the nucleus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Rabbit polyclonal antibodies against Sen2p, Sen54p, Sec63p, Hht1p, Pom152p, and Nsp1p were raised using the corresponding recombinant proteins expressed in Escherichia coli. In the case of anti-Sen2p antibodies, the SEN2 open reading frame (ORF; see below) was subcloned into pET21d (Novagen, Madison, WI) to express Sen2p-His6. The fusion protein was purified from the inclusion body by preparative SDS-PAGE and electro-elution and used for immunization. For preparing antigen-agarose resin, the GSTSen2p(1–219)-His6 fusion protein was expressed in E. coli, purified with Ni-NTA agarose (Qiagen, Tokyo, Japan) under denaturing conditions, and immobilized on Affi-Gel 15 (Bio-Rad, Hercules, CA). A crude IgG fraction prepared by ammonium sulfate precipitation from anti-Sen2p antisera was applied to the Sen2p-agarose. Anti-Sen2p antibodies were eluted with 0.1 M glycine-HCl, pH 2.0, after extensive wash. The eluate was desalted, rechromatographed, adjusted to TBS + 1% BSA, and passed through yeast lysate-agarose, where yeast total lysate extracted with SDS was immobilized on Affi-Gel 15. Affinity-purified anti-Sen54p antibodies were prepared and purified essentially by similar procedures as above with the Sen54p(1–432)-His6 fusion protein as an antigen. The affinity-purified anti-Sen2p and anti-Sen54p antibodies recognized single bands corresponding to their antigens in Western blotting in Figure 1, A and B and 4B. Rabbit antiprotein A antibodies were purchased from Biogenesis (Poole, Dorset, UK) and were passed through yeast lysate-agarose to remove contaminated antiyeast protein antibodies before use. An mAb against yeast Por2p and Alexa 488–and Alexa 546–labeled secondary antibodies were purchased from Molecular Probes (Eugene, OR). Rhodamine-labeled antibodies were from Boehringer Mannheim (Mannheim, Germany). Cy5-labeled secondary antibodies were from Amersham Biosciences (Tokyo, Japan).
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-32P. For FISH, either FITC or Cy5 was conjugated to the
probes by the manufacturer.
Plasmids
The multicopy vectors pYO324 (2µTRP1) and pYO326
(2µURA3) were a gift from Dr. Y. Ohya, University of
Tokyo (Qadota et al.,
1992). A 2.47-kb SacI-SpeI fragment including
the SEN2 gene was amplified by PCR and was subcloned into pRS316,
pRS314, and pYO326 to yield pTYSC017, pCOSC05, and pYU042, respectively. A
4.13-kb SacI-KpnI fragment including the SEN54 gene
was amplified by PCR and was subcloned into pRS316, pRS314, and pYO324,
yielding pTYSC155, p314–54, and pTYSC161, respectively. To construct
FLAG3 or protein A fusion plasmids, a 0.58-kb fragment containing
ADH1 terminator and HIS3 gene from Candida glabrata
(Sakumoto et al.,
1999) were inserted into pBluescript SK(–) in this order. A
90-base pair DNA fragment encoding three tandem FLAG epitopes (DYKDDDDKRP) or
a 710-base pair fragment encoding the IgG-binding domain of protein A from
pRIT2T were inserted in the plasmid to yield pTYE247 and pTYE248,
respectively. To construct p70N-54 with TOM70N-SEN54, the
SEN54 promoter region (0.53 kb), a TOM70 fragment encoding
the first 61 amino acids (0.19 kb) and the SEN54 ORF (1.53 kb) were
amplified, conjugated with appropriate restriction sites by PCR, and assembled
on pBluescript SK(–) in this order. A 0.54-kb
MluI-BglII fragment from p314–54, containing the
5' portion of SEN54 gene, was replaced with that of the above
plasmid to yield p70N-54. The sen54200-232 and
sen54275-313 deletion mutants were constructed by
oligonucleotide-directed mutagenesis of p314–54, using the
oligonucleotides, 5'-GAAACCACTAAACAGCGATTCTTGATTGCTGGGTTTTAG-3'
and 5'-ATTGTAAACGAAAACTTGGAATTTTTTATTCTTTGGTACAGC-3' to yield
p314-200 and p314-275, respectively. The 0.54-kb
MluI-BglII fragment of the two plasmids was replaced with
that of p70N-54 to yield p70N-200 and p70N-275. All of the PCR
fragments were confirmed by DNA sequencing.
Yeast Strains
The yeast strains used in this study are listed in
Table 1. Standard yeast genetic
techniques for the construction of these strains were as described
(Guthrie and Fink, 1991).
Wild-type diploid and haploid strains were W303 or its derivatives. The
diploid strains TYSC257 (SEN54/sen54::HIS3) and
YUSC025 (SEN2/sen2::LEU2) and the
los1::URA3 haploid strains were constructed by one-step gene
replacement (Guthrie and Fink,
1991). For construction of haploid strains with FLAG3-
or protein A-tagged genes, DNA fragments harboring tag sequences,
ADH1 terminator and CgHIS3, were amplified with 60-base pair
tabs homologous to target genes from pTYE247 and pTYE248, respectively, and
integrated into the 3'-end of SEN2 or SEN54 loci of a
wild-type haploid, TYSC188, as described
(Schneider et al.,
1996). To construct the sen2-3 strain, SEN2 on
pTYSC017 was mutagenized to sen2-3 by oligonucleotide-directed
mutagenesis with the oligonucleotide,
5'-TATTATATAAGAGAGAGCCACCATTTCAAC-3'. A haploid strain
whose chromosomal disruption of SEN2 was complemented by this
sen2-3 plasmid was constructed with YUSC025 by tetrad dissection. For
the new sen2 ts selection, pCOSC05 was mutagenized with
hydroxylamine, and ts alleles were isolated by the plasmid shuffling method
with the haploid COSC04–2 (sen2::LEU2/pTYSC017) as the
recipient. Yeast clones that lost pTYSC017 and showed temperature-sensitive
growth were selected on 5'-FOA medium. Strains with various mutant
sen54 genes were also constructed by plasmid shuffling with a haploid
TYSC322 (sen54::HIS3/pTYSC155), which was derived from TYSC257
by tetrad dissection.
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Cell Biological Techniques
Immunofluorescence of yeast cells was done as described
(Guthrie and Fink, 1991).
Fluorescent images were acquired with an Olympus IX70 microscope equipped with
a MicroMax cooled CCD camera (Roper Scientific, Tucson, AZ), and were analyzed
by IP Lab (Scanalytics, Inc., Billerica, MA).
For the overall cell fractionation, yeast cells grown in appropriate media were converted into spheroplasts and were disrupted by agitation with glass beads for 30 s in lysis buffer (20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 10 mM spermidine-HCl, 0.5 mM DTT, 200 mM (NH4)2SO4, 10% sucrose, and protease inhibitor cocktail). After the cell debris was removed, the lysate was centrifuged at 10,000 x g for 20 min to obtain the medium speed pellet (MSP) fraction. The MSP fraction was subjected to a 20–80% sucrose gradient and was centrifuged at 100,000 x g for 36 h at 4°C with a swing-out rotor (RPS27–2, Hitachi, Tokyo, Japan). The gradient was recovered from the bottom. The recovery of organelle markers was assayed by quantitative Western blotting with specific antibodies.
Mitochondria were prepared as described
(Lewin et al., 1990).
Briefly, yeast cells grown in a medium with lactate as a carbon source were
converted into spheroplasts and disrupted in 5 mM MES-KOH, pH 6.0, 0.5 mM EDTA
and 0.6 M sorbitol with a Dounce homogenizer. After the cell debris was
removed, the organelle fraction was recovered by centrifugation at 27,000
x g for 10 min. The pellet was suspended in a buffer with 0.24
M sucrose, subjected to a gradient consisting of layers of 17, 25, and 37%
Nycodenz and then centrifuged at 100,000 x g for 2 h. Fractions
were recovered from the bottom.
Submitochondrial fractionation was carried out as described
(Jascur, 1991). Intact
mitochondria suspended in a buffer with 0.6 M sorbitol were diluted by 10-fold
with 20 mM HEPES-KOH, pH 7.4, and were sonicated to produce mitochondrial
membrane vesicles. After the removal of the unlysed mitochondria by
centrifugation at 30,000 x g for 20 min, the supernatant was
centrifuged at 100,000 x g for 1 h to collect the vesicles. The
pellet was suspended and subjected to a 0.85–1.6 M linear sucrose
gradient. After centrifugation at 100,000 x g for 20 h, the
gradient was collected from the bottom.
Yeast nuclei were prepared by PVP-sucrose gradient centrifugation as
described (Rout and Kilmartin,
1990). Briefly, yeast cells grown in YPD were converted to
spheroplasts. The spheroplasts were suspended as 1 x 109
spheroplasts/ml in PVP solution (20 mM K-phosphate, pH 6.5, 0.5 mM
MgCl2, 8%wt/vol polyvinylpyrrolidone [PVP-40] supplemented with
protease inhibitors cocktail). After addition of final 0.02%wt/vol Triton
X-100, the suspension was homogenized with a Dounce homogenizer and then mixed
with an equal volume of 0.6 M sucrose in PVP solution. Crude membranes were
recovered after centrifugation at 10,000 x g for 10 min, and
suspended in 1.7 M sucrose in PVP Solution. The suspension was loaded onto a
2.01M/2.10 M/2.30 M sucrose discontinuous gradient in PVP solution and
centrifuged at 100,000 x g for 4 h. Nuclei banded at 2.10
M/2.30 M sucrose interface were recovered.
Northern Analysis
Total small RNAs were prepared from yeast cells by the hot phenol method
with GTE (100 mM Tris-HCl, pH, 7.6, 10 mM EDTA, 4 M guanidine thiocyanate) as
a lysis buffer (Guthrie and Fink,
1991). Total RNA samples were separated on 10% polyacrylamide gel
with 7 M urea, transferred to Hybond N+ (Amersham Biosciences) by
electric blotting, and then hybridized with appropriate oligonucleotide probes
terminally labeled with [-32P]ATP. The radioactivity on the
membranes was detected with Imaging Plate (Fuji Film, Tokyo, Japan) and Storm
860 Image Analyzer (Molecular Dynamics, Sunnyvale, CA).
tRNA Splicing Endonuclease Assay
For analyzing the tRNA endonuclease activity, cell extracts were prepared
as described (Winey and Culbertson,
1988). Endonuclease assays were performed with an end-matured,
unspliced form of pre-tRNA-PheGAA labeled with
[
-32P]ATP as a substrate in 10 µl of reaction mixture (40
mM Tris-HCl, pH 8.0, 10% wt/vol glycerol, 0.5 mM EDTA, 20 mM
(NH4)2SO4, 4 mM spermidine-HCl, 0.2% wt/vol
Triton X-100) at 30°C (Peebles et
al., 1983). Products were analyzed on a 10% polyacrylamide
gel with 6 M urea. The radioactivity in the gel was detected as above.
FISH Analysis
FISH was performed essentially as described
(Sarkar and Hopper, 1998) with
some modifications. Hybridization was performed in 4x SSC, 10% dextran
sulfate, 0.2% BSA, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml E. coli
tRNA, 0.5 U/µl RNasein, and 0.1 pmol/µl an FITC- or Cy5-labeled probe at
42°C. Stringent washes were done four times in 4x SSC for 15 min at
42°C. After the final wash, DNA was stained with 0.3 µg/ml
4',6-diamidino-2-phenylindole (DAPI) in 4x SSC for 5 min.
Fluorescent images were acquired with an Olympus IX70 microscope equipped with
a MicroMax cooled CCD camera (Roper Scientific), and were analyzed by IP Lab
software (Scanalytics, Inc.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), most of the Sen2p signals were distributed as
discrete dots along tubular structures that were distinct from the nucleus
(Figure 1A, WT). These
structures were identified as mitochondria by costaining with a mAb against
Por2p, a mitochondrial marker. Only minor, if any, signals were detected in
other regions of the cell, including the nucleus. Similar images were obtained
when Sen54p, another subunit of the Sen complex, was detected with its
antibodies despite the fact that Sen54p has a typical NLS-like sequence,
402KKKR406K (Figure
1B, WT). The mitochondrial signal by the antibodies is specific to
Sen54p, because a deletion mutation of its mitochondrial localization domain
altered the signal distribution (Figure
5C; see below). The punctate signals of Sen2p and Sen54p on the
mitochondria partially result from the fact that only a small number of Sen
complexes (
100 molecules) exist in a cell
(Trotta et al.,
1997). It seems that these few molecules are not enough for
uniform signals on the mitochondria. In fact, when overproduced from a
multicopy plasmid, Sen2p and Sen54p were more evenly distributed along the
mitochondria, and their signals were enhanced
(Figure 1, A and B, OP). By
Western blotting, the Sen2p overproducer and Sen54p overproducer expressed 13-
to 17-fold Sen2p and 10- to 16-fold Sen54p, respectively. Preimmune sera of
the anti-Sen2p or anti-Sen54p antibodies did not produce such mitochondrial
staining in the immunofluorescence (our unpublished results). These
observations were not specific to our strain background. We obtained
essentially the same results with several wild-type strains, including 20-B12
that was used in Peebles et al.
(1983) (our unpublished
results).
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To confirm the localization of Sen2p and Sen54p on mitochondria, we monitored localization of Sen2p-protein A and Sen54p-protein A fusion proteins that were expressed from chromosomes with their authentic promoters. These strains grew normally and expressed only full-size fusion proteins (Figure 1C, right most panel), indicating full functions of the fusion proteins. Anti-protein A antibodies again revealed colocalization of these protein A fusions with Por2p (Figure 1C). No signal was detected in their parental strain TYSC188 (Figure 1C, no-protein A) and in experiments without anti-protein A antibodies (our unpublished results). We confirmed that protein A fusions are not recognized by the anti-Por2p antibody (mouse IgG1) and the secondary antibodies (from goat) under these conditions. These results indicate that Sen2p and Sen54p are localized mainly to the mitochondria and also suggest that the two subunits can be present on the mitochondria by themselves.
To further establish the mitochondrial localization of the tRNA splicing
endonuclease activity in wild-type cells, we performed subcellular
fractionations. Although the endonuclease was shown to be associated with
membranes, the distribution of its activity was not compared with various
organelle markers (Peebles et
al., 1983). By differential centrifugation, Sen2p and Sen54p
in wild-type cells were mainly recovered in an MSP fraction, a pellet from a
10,000 x g centrifugation. When the MSP was resolved on a
20–80% wt/vol sucrose density gradient, Sen2p and Sen54p were
distributed with a major peak around fraction 11 and a minor peak around
fraction 17 (Figure 2A). These
patterns resemble those of the mitochondrial markers, Tim23p and Tom40p
(Vestweber et al.,
1989; Emtage and Jensen,
1993), but are clearly different from those of the nuclear
markers, histone H3 (Hht1p), Nsp1p and Pom152p
(Hurt, 1990;
Wozniak et al., 1994;
Kumar et al., 2002),
and an ER marker, Sec63p (Feldheim et
al., 1992; Figure
2A; our unpublished results). We then assayed the tRNA
endonuclease activity in each fraction with end-matured
pre-tRNA-PheGAA as a substrate. The endonuclease activity that
cleaves the pre-tRNA into two exons and an intron also showed a distribution
similar to that of the mitochondrial markers, with a major peak around
fraction 11 with a shoulder around fraction 17
(Figure 2, A and B).
Cofractionation of the endonuclease with the mitochondria was also observed in
different gradient systems with Nycodenz (our unpublished results).
To confirm that the detected endonuclease activity arose from the Sen
complex, we carried out a similar fractionation with sen2-3 mutant
cells. The sen2-3 cells cleave the intron-3'-exon junction
normally, but cleave the 5'-exon-intron junction inefficiently,
resulting in the accumulation of a 5'-exon-intron 2/3 molecule
(Ho et al., 1990).
The pre-tRNA cleavage activity of sen2-3 cells, mainly detected in
the MSP fraction, now processed the pre-tRNA to the 2/3 molecule and the
3'-exon (Figure 2B). All
of the fractions throughout the gradient of sen2-3 membranes
processed the pre-tRNA into the same molecules
(Figure 2B, lower). Therefore,
the endonuclease activity we detected indeed came from the Sen complex,
indicating that the majority of the tRNA endonuclease activity is associated
with the mitochondria.
Because resolution of the overall fractionation is not sufficient for
complete separation of mitochondria and the nucleus, we performed
organelle-specific fractionations. First, we prepared intact mitochondria from
wild-type cells by Nycodenz gradient centrifugation
(Lewin et al., 1990).
Sen2p and Sen54p were enriched in the mitochondrial fraction with purity
similar to that of the mitochondrial markers, Tom70p
(Lithgow et al.,
1994) and Tim23p, whereas the nuclear markers, Hht1p and Nsp1p,
peaked in different fractions (Figure
2C, left). Then, we prepared nuclei by PVP-sucrose density
gradient centrifugation (Rout and
Kilmartin, 1990). Hhtp and Nsp1p were enriched in the nuclear
fraction more than 10-fold. In this fraction, Sen2p and Sen54p were somewhat
contaminated but their levels were similar to those of mitochondrial markers
(Figure 2C, right). These
results again support the idea that the majority of tRNA splicing endonuclease
is localized to the mitochondria and no significant pool of the enzyme exists
in the nucleus.
Next, we investigated the localization of Sen2p and Sen54p within the mitochondria. Mitochondrial membrane vesicles were prepared and separated into outer mitochondrial membrane (OM) vesicles and inner mitochondrial membrane (IM) vesicles with a sucrose density gradient. The sedimentation patterns of Sen2p (Figure 3A) and Sen54p (our unpublished results) resembled that of Tom70p (OM), but differed from that of Tim23p (IM). The contaminating NE, represented by Nsp1p, was purified away from the OM. Therefore, the Sen complex is associated with the mitochondrial OM. We then treated intact mitochondria with proteinase K to see whether these subunits were exposed on the cytosolic surface of the mitochondria. The two subunits were degraded by proteinase K (Figure 3B). Tim23p, an IM protein with a domain exposed to the intermembrane space, was protected from the digestion, whereas an OM protein, Tom70p, was degraded, indicating that the OM was intact. All of the proteins were digested by the protease in the presence of Triton X-100. This implies that Sen2p and Sen54p are exposed to the cytosol.
The endonuclease was regarded as an integral membrane protein, because the
release of the activity from membranes requires detergent and Sen2p has a
hydrophobic stretch (Peebles et
al., 1983; Trotta et
al., 1997). We then analyzed the membrane interactions of
Sen2p and Sen54p. They were released from the mitochondrial membrane with 0.2
M Na2CO3, which can extract peripheral membrane proteins
and with Triton X-100 in the presence of 1 M NaCl. The integral membrane
proteins Tom70p and Tim23p were solubilized only in the presence of the
detergent (Figure 3C). These
results indicate that Sen2p and Sen54p are peripheral membrane proteins. The
crystal structure of a soluble archaeal tRNA endonuclease, a homologue of
Sen2p, also suggests that the putative transmembrane domain of Sen2p
(225–243) is embedded in the interior of the protein
(Li et al., 1998).
Finally, we tested whether the endonuclease activity itself was extracted with
0.2 M Na2CO3, which seemed harsh for the enzyme. In
fact, some of the activity was solubilized under these conditions
(Figure 3D), whereas the total
activity was reduced to 67% of its original value. Therefore, we concluded
that the yeast tRNA endonuclease is peripherally associated with the cytosolic
surface of the mitochondrial OM.
A Sen54p Fusion Protein Artificially Fixed on Mitochondria Is
Functional
Although the above results indicate that the majority of Sen2p, Sen54p and
the tRNA endonuclease activity are associated with the mitochondria, it is
still possible that the endonuclease shuttles between the mitochondria and the
nucleus dynamically and carries out the tRNA splicing in the nucleus. We thus
investigated this possibility. The localization of Sen2p and Sen54p was not
altered in several nuclear transport mutants (our unpublished results).
However, it is difficult to rule out the nuclear-cytoplasmic shuttling by the
analysis of transport mutants, because some proteins utilize multiple
receptors for their import and others do not even require the Ran gradient
across the NE for their transport (Rout
et al., 1997;
Takizawa et al.,
1999). Therefore, we adopted a different approach. We constructed
a fusion gene encoding Tom70N-Sen54p, in which the entire Sen54p is fused to
the C termini of the first 61 residues of Tom70p (Tom70N). This region of
Tom70p is sufficient to anchor a nonmitochondrial protein to the mitochondrial
OM as an integral membrane protein (Nakai
et al., 1989), and thus Tom70N-Sen54p is expected to
behave as an integral membrane protein. It has been demonstrated that the NLS
for soluble proteins cannot deliver integral membrane proteins into the
nucleus (Soullman and Worman,
1995). Therefore, this fusion protein is not expected to shuttle
between the mitochondria and the nucleus and should be sequestered from the
nucleus.
We put the fusion ORF under the SEN54 promoter on a low copy
plasmid and tested whether the fusion gene could replace the authentic
SEN54 by tetrad analysis. Because SEN54 is essential for
yeast growth, a diploid strain with one disrupted copy of SEN54 on
its chromosomes produced two viable (SEN54) and two dead spores
(sen54) (Figure
4A, Vec). However, the diploid strain harboring a low-copy plasmid
with TOM70N-SEN54 produced three or four viable spores,
resembling dissections with the authentic SEN54 on a plasmid
(Figure 4A, 54 and 70N-54). No
growth defects were observed in the viable spores (our unpublished results).
Genetic markers indicated that all of the viable haploids with a disrupted
copy of SEN54 on their chromosome had the
TOM70N-SEN54 fusion gene on the plasmid. These results
suggest that the fusion gene can functionally replace SEN54.
Western blotting revealed that only a full-size Tom70NSen54p, but no degradation product, was present in total cell extracts (Figure 4B). Tom70N-Sen54p, like Sen54p, was colocalized with the mitochondrial marker, Por2p (Figure 4B). We then analyzed the interaction of the fusion protein with membranes. In contrast to Sen54p, Tom70N-Sen54p was not extracted from the crude membranes with Na2CO3, indicating that it behaves as an integral membrane protein (Figure 4C). We next examined the pre-tRNA accumulation in vivo and the endonuclease activity in vitro in the TOM70N-SEN54 cells. Northern blotting demonstrated that the amounts of unspliced precursors of tRNA-IleUAU, tRNALeuCAA, and tRNA-ProUGG in the TOM70N-SEN54 cells were similar to those of the wild-type cells (Figure 4D). We then prepared crude extracts from the TOM70N-SEN54 and wild-type cells and assayed the tRNA endonuclease activity in vitro with pre-tRNA-PheGAA as a substrate. There was essentially no difference in the endonuclease activity between the two strains (Figure 4E). Therefore, Sen54p can function even when it is artificially fixed on the mitochondria, providing evidence against the nuclear-cytoplasmic shuttling of the Sen complex.
sen54 Mutants Defective in Mitochondrial Localization Are Not
Fully Functional
Next, we asked whether the mitochondrial localization of Sen54p is required
for its function. First, we identified the mitochondrial localization signal
of Sen54p by deletion analyses. Its region including amino acids 200–313
was sufficient for targeting GFP to the mitochondria (unpublished results).
Then, we constructed TRP1 low-copy plasmids harboring SEN54
derivatives with partial deletions of this region:
sen54200-232 (p314-200) and
sen54275-313 (p314-275)
(Figure 5A). We introduced
these plasmids into a haploid strain whose chromosomal SEN54
disruption is complemented by the authentic SEN54 on a URA3
plasmid. Cells losing the URA3 plasmid were selected on a
5'-FOA plate. Cells with p314-275 could not grow on the
5'-FOA plate, indicating that sen54275-313
cannot complement sen54
(Figure 5B, top).
On the other hand, sen54200-232 cells grew on the
5'-FOA plate and showed a ts phenotype. At 37°C,
sen54200-232 cells accumulated unspliced pre-tRNAs
(our unpublished results). We observed similar but weak splicing defects in
another partial deletion of the SEN54 mitochondria targeting domain,
sen54233–246 (our unpublished results).
Sen54200-232p was no longer localized to the mitochondria, but was
distributed throughout the cytoplasm even at the permissive temperature
(Figure 5C). There are some
Sen54p signals in the nuclear regions of the mutant cells. The deletion
mutation might result in exposure of the NLS-like sequence,
402KKKR406K, in Sen54p through alteration of its overall
structure. If the growth defect comes from abnormal localization of Sen54p,
regaining the mitochondrial localization by introducing an ectopic
localization signal will suppress the defect. Fusion of the TOM70N to
the 5'-terminus of the sen54200-232 ORF
resulted in the recovery of the growth at 37°C and of the mitochondrial
localization of the mutant protein (Figure
5, B, bottom, and C). The in vitro endonuclease activity at
30°C of sen54200-232 extracts was reduced to 29%
of the wild-type extracts, but the activity of
TOM70N-sen54200-232 extracts was recovered
to 68%, indicating that the mitochondrial localization of Sen54p correlates
with the total endonuclease activity.
Mutations in a localization signal of a protein are expected not to affect
the total activity of the protein if its signal domain and catalytic domain
are separated. However, in the case of yeast tRNA endonuclease, a
multi-subunit enzyme that has more than two subunits with independent
localization information, failure in mitochondrial targeting of Sen54p alone
will cause inefficient complex assembly on the surface of mitochondria,
leading to a decrease in the total endonuclease activity. Indeed,
Figure 1, A and B, demonstrate
that Sen54p and Sen2p can be targeted to the mitochondria independently.
Furthermore, the localization of Sen2p was not affected by
sen54200-232 mutation
(Figure 5D), indicating that
only a part of Sen54p was colocalized with Sen2p. Tom70N-Sen54275-313p
was not functional (Figure 5B), implying that the 275-313 region has a role other than in mitochondrial
localization. These results indicate that the defects of the
sen54200-232 mutant come from its mislocalization
from the mitochondria and that the mitochondrial localization of Sen54p is
required for the assembly and/or enzymatic activity of tRNA endonuclease.
Endonuclease-deficient Cells Accumulate pre-tRNAs in the Cytosol
If the tRNA splicing endonuclease functions on the mitochondrial surface,
the unspliced pre-tRNAs must be exported from the nucleus. We thus analyzed
the localization of unspliced pre-tRNAs in endonuclease deficient mutants by
FISH. We focused on Sen2p, one of the catalytic subunits of the enzyme.
Because the sen2-3 mutation is leaky and not conditional
(Ho et al., 1990), we
screened new sen2 ts alleles and obtained one allele,
sen2-41, that showed a clear ts growth
(Figure 6A). Northern blotting
showed that sen2-41 cells accumulated end-matured unspliced forms of
tRNA-IleUAU, tRNA-LeuCAA, and tRNA-ProUGG
during a 4-h incubation at 37°C (Figure
6B). 5'-exon-intron 2/3 molecules and introns were
apparently not accumulated in this mutant (our unpublished results). Even at
23°C, the mutant cells contained higher amounts of the pre-tRNAs than
wild-type cells. Next, we tested the tRNA endonuclease activity in vitro. The
sen2-41 extract prepared from the mutant cells grown at 23°C had
significantly lower endonuclease activity, when tested at 30°C
(Figure 6C). The extract was
also inactive at both 23 and 37°C (our unpublished results). A reaction
product corresponding to a 5'-exon-intron 2/3 molecule was detected in
the gel. Therefore, the mutant has a primary defect in the cleavage of the
5'-exon-intron junction of pretRNAs in vitro.
|
Using the sen2-41 mutant, we analyzed the localization of
tRNA-IleUAU by FISH with two probes: one recognizing both the
precursor and mature forms of the tRNA and the other against its intron.
First, we monitored the time course of pre-tRNA accumulation after shift to
the restrictive temperature. As reported previously
(Sarkar and Hopper, 1998;
Grosshans et al.,
2000), in wild-type cells, unspliced pretRNA-IleUAU was
detected mainly in the nucleus at 23°C. Its localization was not changed
by up to 4-h incubation at 37°C (Figure
7A, wt, left column). We usually saw a transient decrease in the
signal intensity within 1 h after the shift. This transient decrease was also
detected by Northern blotting (Figure
7B). In the sen2-41 cells, the unspliced
pre-tRNAIleUAU localization at 23°C rather resembled that of
the wild-type cells. The pre-tRNA signal decreased transiently within 1 h
after the shift to 37°C, as was the case of the wild-type cells. The
pre-tRNA then accumulated gradually from 2 h after the shift
(Figure 7A, sen2, left
column). The increase in the cytosolic signal was more prominent than that in
the nucleus during this period. Within 3 h, most of the cells accumulated
similar levels of the pre-tRNA, in both the cytosol and the nucleus. The
increase of the cytosolic pre-tRNA in the mutant cells monitored by FISH
correlates well with the total accumulation of the pre-tRNA monitored by
Northern blotting (Figure 7B).
Experiments with intronspecific probes for tRNA-TrpCCA,
tRNA-LeuCAA, and tRNAProUGG gave similar results (our
unpublished results). These results indicate that the unspliced pre-tRNAs are
exported to and accumulated in the cytosol in the absence of the splicing
endonuclease activity. The fact that an extremely high accumulation of the
pre-tRNA in the nucleus was not observed argues against the possibility that
the cytosolic signal is a mere leakage from the nucleus where high amounts of
pre-tRNAs accumulate because of the lack of the splicing activity. The
mutation did not affect the localization of total tRNA-IleUAU
(Figure 8, n and t), mature
tRNATrpCCA, mature tRNA-LeuCAA, and the intron-less
tRNAs, tRNA-GlyGCC, and tRNA-GluUUC (our unpublished
results). The integrity of the NE and intranuclear structures, like the
nucleolus, seems to be intact in the sen2-41 cells, because the
locations of U14 snoRNA (Figure
7C), U6 snRNA (our unpublished results), and mRNA
(Figure 7D) in the mutant cells
were indistinguishable from those in the wild-type cells.
|
|
Next, we analyzed the localization of tRNA-IleUAU in a
los1 sen2-41 double mutant to assess the effects of a mutation
in the tRNA export machinery. los1 cells, which lack the yeast
exportin-t homologue Los1p, accumulate both the pre-tRNA and the mature tRNA
in the nucleus as expected (Sarkar and
Hopper, 1998; Figure 8,
d–f and j–l). The nuclear pool of the pre-tRNA in the
los1 cells was much larger than that in the sen2-41
cells. In the los1 sen2-41 cells, the pretRNA accumulated in
the nucleus, like the los1 cells, but a considerable amount of
the pre-tRNA was present in the cytosol, like the sen2-41 cells
(Figure 8, v–x). Similar
results were obtained with intron-specific probes against
tRNATrpCCA, tRNA-LeuCAA, and tRNA-ProUGG (our
unpublished results). These results further indicate that the mere
accumulation of the pre-tRNAs in the nucleus does not result in the leakage of
the pre-tRNAs to the cytosol. The simplest interpretation of the experiments
described above is that the pre-tRNAs are actively exported to the cytosol, in
a manner that is partly dependent on Los1p. The results are consistent with
the idea that tRNA splicing occurs on the mitochondria.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) suggested that the
yeast tRNA splicing endonuclease was an integral membrane protein of the inner
NE. However, the biochemical and cytological analyses in the present study
revealed that the yeast endonuclease mainly localizes to the mitochondria, and
only a small amount, if any, of the enzyme exists in the nucleus. In
Xenopus oocytes, the tRNA endonuclease activity was mainly detected
in the nucleoplasm (DeRobertis et
al., 1981) and is thought to act in the nucleus in vivo
(Melton et al.,
1980). This raises the possibility that the primary location of
this enzyme differs in different organisms and/or cell types. The present
mutant analysis in yeast further suggests that the mitochondrial localization
of the tRNA endonuclease has a positive role in tRNA splicing. Finally, we
obtained pieces of evidence that support pre-tRNA export from the nucleus.
These results apparently do not fit the currently accepted model that tRNA
splicing occurs in the nucleus and only mature tRNAs are exported to the
cytoplasm.
Although we demonstrated that large portions of Sen2p, Sen54p, and the
endonuclease activity are localized on the mitochondria, there are still two
explanations for these observations from the view of nuclear pre-tRNA
splicing. One possibility is nuclear-cytoplasmic shuttling of the
endonuclease. However, the Sen54p derivatives inserted in the OM are fully
functional. Disintegration of integral membrane proteins from membranes would
require special machinery that does not seem to function in the usual
nuclear-cytoplasmic transport. Therefore, a large part of Tom70N-Sen54p should
exist and function on the OM. This fact suggests that the above possibility is
less likely. The other explanation is that a minor nuclear pool of the
endonuclease is responsible for the tRNA splicing. The Sen complex may
assemble on the mitochondrial surface and be stored there until it leaves for
the nucleus. It may also be possible that the mitochondrial Sen complex has
another function instead of tRNA splicing. There are many examples where one
protein localizes at two distinct cellular compartments and each portion of
the enzyme has different functions
(Danpure, 1995). Even in the
case of RNA processing, RNase P and RNase MRP share most of their protein
subunits but fill distinct roles of pre-tRNA end-processing in the nucleus and
5.8S rRNA processing in the mitochondria, respectively
(Chamberlain et al.,
1998; Gold et al.,
1989). The Sen complex might be another example of this case. In
fact, it is logically difficult to prove that no tRNA endonuclease exists in
the nucleus. At least, our various fractionation and immunofluorescence
analyses did not reveal a significant pool of the enzyme in the nucleus. In
TOM70N-SEN54 cells, a larger amount of the Sen54p moiety should be
trapped on the mitochondria than in wild-type cells. Therefore, the nuclear
pool of the enzyme should be quite small. Because only 100 molecules of
the endonuclease exist in a yeast cell
(Trotta et al.,
1997), these observations suggest that, for tRNA splicing in the
nucleus, only several molecules of the enzyme in a nucleus would have to
provide the entire function in wild-type cells that grow normally. On the
other hand, alteration of the mitochondrial localization of Sen54p by the
partial deletion of its localization signal compromises normal growth and tRNA
splicing activity. Regain of mitochondrial localization by addition of an
unrelated targeting signal is enough to restore the phenotypes. Therefore,
these observations favor the idea that the mitochondrial pool of Sen54p
contributes to the tRNA splicing in yeast cells.
In the presence of our new observations, how can we explain the nuclear
accumulation of end-matured, unspliced pre-tRNAs in the mutants defective in
tRNA export? As mentioned before, several observations do not support
"splicing-export coupling model" based on the nuclear splicing.
The existence of a proofreading step by RS indicates that splicing,
proofreading, and export occur sequentially in this order
(Lund and Dahlberg, 1998;
Sarkar et al., 1999;
Grosshans et al.,
2000). Because excess amounts of mature tRNAs do not inhibit tRNA
splicing in vitro, product inhibition by mature tRNAs accumulated in the
nucleus is unlikely (Peebles et
al., 1979). In fact, when the aminoacylation of a certain
tRNA is blocked, only its mature form, but not its precursor, is accumulated
in the nucleus, indicating that such product inhibition does not occur even in
vivo (Azad et al.,
2001). The discovery of intranuclear translation predicts that the
nuclear aminoacyl-tRNAs have a vital role in this process, indicating that
there is an exchangeable pool of mature tRNAs in the nucleus before export
(Iborra et al.,
2001). Finally, we showed that sen2-41 cells accumulate
pretRNAs in the cytosol and that this was not due merely to leakage from the
nucleus. The nuclear splicing model predicted that endonuclease-deficient
mutants would accumulate unspliced pre-tRNAs in the nucleus; otherwise,
sen2-41 should be a special mutant that compromised nuclear tethering
of unspliced pre-tRNAs. A SEN54-depletion strain also accumulated
pre-tRNAs in the cytosol (Tanaka, Endo, and Yoshihisa, unpublished results),
suggesting that the cytosolic accumulation of pre-tRNAs is a general phenotype
in sen mutants. Because the amount of the endonuclease in the nucleus
seems to be very small, if the endonuclease plays a role in retaining
pre-tRNAs in the nucleus, it would have to act catalytically. Therefore,
several conditions are required to understand ours and other's observations
from the view of the nuclear splicing.
On the contrary, if we accept that the endonuclease on the mitochondria is
physiologically active, these observations can be easily explained in the
following manner. The pretRNAs are exported to the cytoplasm for their
splicing on the mitochondria, and this export is the rate-limiting step for
their splicing; therefore, the export mutations prevent the pre-tRNAs from
access to the endonuclease. Several parallel pathways for tRNA export have
been reported (Hellmuth et al.,
1998; Azad et al.,
2001; Feng and Hopper,
2002). These pathways may be divided into two classes. One is
specific for fully matured and functional tRNAs, and is governed by RSs,
eEF-1, etc. The other can export the pre-tRNAs and partly depends on
Los1p. Indeed, the los1 mutation causes the nuclear accumulation of
the mature form of introncontaining tRNAs, but not that of some intronless
tRNAs (Grosshans et al.,
2000). Although we have not definitively proven that pre-tRNAs are
exported to the cytosol to gain access to the mitochondrial endonuclease, the
mitochondrial splicing model should be considered as a probable working
hypothesis.
If pre-tRNAs are cleaved on the mitochondrial surface, then a question may
arise as to the fate of the resulting tRNA exons. The exons may come back to
the nucleus to be ligated by the yeast tRNA ligase (Rlg1p) localized there
(Clark and Abelson, 1987), and
the ligated tRNAs may be exported again to the cytosol. Alternatively, Rlg1p
may shuttle between the nucleus and the cytosol to mediate tRNA ligation in
the cytosol. Rüegsegger et al. reported that the yeast
HAC1 pre-mRNA is spliced in the cytosol by a nonconventional
mechanism similar to that of the pre-tRNAs. HAC1 exons are indeed
joined by Rlg1p, suggesting that a fraction of Rlg1p functions in the cytosol
(Sidrauski et al.,
1996; Rüegsegger et
al., 2001). In any case, if intranuclear translation occurs
in yeast as in the case of mammalian cells, a fraction of the mature tRNAs
must be retained in or sent back to the nucleus.
Another fact that may not be consistent with the mitochondrial splicing
model is that splicing and end-processing of tRNAs are not completely ordered
to each other (O'Connor and Peebles,
1991). Because the RNA subunit of RNase P, the RPR1 gene
product, was found to be associated with the nucleolus
(Bertrand et al.,
1998), end-immature spliced pre-tRNAs must go back to this nuclear
compartment for their end-processing. As mentioned above, our data suggest
existence of some mechanism that operates for nuclear import of tRNA
molecules. Such mechanism may support the pathway where the splicing precedes
the endmaturation. At least, we mainly detected end-mature precursors of
tRNA–IleUAU, tRNA-LeuCAA, and
tRNA-ProUGG in sen2-41 mutant cells, indicating that the
splicing defects do not affect the end-processing in the mutant. On the other
hand, end-immature spliced pre-tRNAs were accumulated in mutants of Sm-like
proteins (Lsm proteins) and a La homologue in both Saccharomyces
cerevisiae and Schizosaccharomyces pombe
(Intine at al., 2002;
Kufel et al., 2002).
Especially, a La derivative that is not efficiently retained in the nucleus
causes accumulation of end-immature spliced pretRNAs, whereas this mutant La
interacts with these end-immature species
(Intine et al.,
2002). Because Lsm and La proteins are thought to interact with
early processing precursors of tRNAs, the end-immature spliced pre-tRNAs
accumulated in these mutants might escape from the normal processing pathway
to be delivered to the cytoplasm. Or the aberrant retention of the
end-immature species in the cytosol may cause inefficient end-processing in
the splicing-first pathway. Most of these results, including ours, were
obtained through Northern blotting. Therefore, further investigation with
pulse-chase experiments will be necessary to reveal exact fates of
incompletely processed species in tRNA biogenesis.
In summary, accumulated findings including ours suggest that, in yeast, the tRNA splicing endonuclease on the mitochondria has positive roles in tRNA biogenesis, although its physiological meaning is still obscure. The tRNA traffic in eukaryotic cells should be carefully reexamined in the view of the unexpected finding that the enzyme is localized on mitochondria. The tRNAs themselves, but not the processing enzymes, may dynamically shuttle between the nucleus and the cytoplasm during and after their maturation.
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ACKNOWLEDGMENTS |
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Footnotes |
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Abbreviations used: 5'-FOA, 5'-fluoroorotic acid; DAPI, 4', 6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; IM, inner mitochondrial membrane; MSP, medium speed pellet; NE, nuclear envelope; NLS, nuclear localization signal; OM, outer mitochondrial membrane; ORF, open reading frame; PVP, polyvinylpyrrolidone; RS, aminoacyl-tRNA synthetase; Sen, splicing endonuclease.
Corresponding author. E-mail address:
tyoshihi{at}biochem.chem.nagoya-u.ac.jp.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, Tom70p;
, Tim23p; 
, Nsp1p; 
, Sen2p. The fraction P represents the
pellet of the gradient. (B) The intact mitochondria were treated with 0.1
mg/ml proteinase K at 4°C for 20 min in the absence or presence of
0.2%wt/vol Triton X-100 and were analyzed by Western blotting. (C) The
mitochondria (T) were extracted with lysis buffer (Buffer), 0.2 M
Na2CO3 (Na2CO3) or lysis buffer
with 1% Triton X-100 + 1 M NaCl (Triton) and were separated into a pellet (P)
and a supernatant (S) by centrifugation at 100,000 x g for 30
min. Each protein was detected with specific antibodies. (D) The mitochondria
were extracted as in C. The supernatants were passed through gel filtration
columns to exchange the buffers to lysis buffer with 1% Triton X-100. The
pellets were suspended in the same buffer. The endonuclease activity in each
fraction was assayed and is represented by the recovery.
) were compared with that
of the endonuclease activity (
