(Received for publication, September 28, 1995; and in revised form, December 5, 1995)
From the
CCA1 codes for mitochondrial, cytosolic, and nuclear ATP(CTP):tRNA nucleotidyltransferase. Studies reported here examine the mechanisms leading to and the consequences of altering the distribution of this important tRNA processing enzyme. We show that the majority of Cca1p-I, translated from the first in-frame ATG, is in mitochondria but surprisingly, there is a small contribution to nuclear and cytosolic tRNA processing by this form as well. The majority of Cca1p-II and Cca1p-III, translated from ATG2 and ATG3, respectively, is in the cytosol but both are also located in the nucleus for processing precursors. Altering the cytosolic/nuclear distribution of Cca1p by fusing the SV40 nuclear localization signal to the 5` end of CCA1 causes a growth defect and results in the accumulation of end-shortened tRNAs in the cytosol. These results suggest an important role for Cca1p in the cytosol of eukaryotes, presumably in the repair of 3` CCA termini. These experiments also demonstrate that individual tRNAs are affected differently by reduced cytosolic nucleotidyltransferase and that cells resuming exponential growth are more severely affected than those continuing exponential growth.
Transfer RNA biosynthesis is compartmentalized in eukaryotics as both nuclear and mitochondrial DNA generally contain tRNA genes. In the instances in which nuclear transcribed tRNAs are imported into mitochondria, it is not clear in which cellular compartment maturation occurs but it is clear that all steps in the biosynthesis of tRNAs coded by mitochondrial DNA occurs in the organelle (for review, see (1) ). Mitochondrial pre-tRNAs are processed to their mature form by steps in which sequences are trimmed from their 5` and 3` ends, bases are modified, and the oligonucleotide CCA is added to the 3` terminus. The order of mitochondrial pre-tRNA processing can vary. Removal of 5` leader sequences from pre-tRNAs need not precede the removal of the 3` trailer sequence and the addition of the end terminal CCA(1, 2) .
In contrast, cytosolic tRNA biosynthesis begins in the nucleus and is completed in the cytosol. The processing of nuclear precursor tRNA to form mature tRNA also requires the trimming of 5` leader and 3` trailer sequences from the primary transcript, the post-transcriptional addition of oligonucleotides CCA to the 3` terminus, base modifications, and in some instances, splicing to remove intervening sequences (for review, see (2) ). Two lines of evidence demonstrate that end maturation, intron splicing, and certain base modifications take place in the nucleus. First, yeast pre-tRNAs microinjected into Xenopus oocyte nuclei are processed(3, 4, 5) . Second, nuclear restricted pre-tRNAs, those tRNAs with intervening sequences, have base modifications and CCA ends(6, 7, 8) . Following maturation in the nucleus, which includes CCA addition, tRNAs are exported to the cytosol where additional base modifications (4, 9) are added. 3` terminal CCA ends turn over in the cytosol(10) .
Multiple locations of tRNA biosynthesis
means that tRNA maturation enzymes with analogous functions are
required in multiple cellular locations. Many of these isozymes are
structurally distinct and are coded by separate genes. However, three
tRNA maturation enzymes have now been identified as sorting isozymes,
enzymes that function in more than one cellular compartment. These
include N,N
-dimethylguanosine-specific
tRNA methyltransferase, Trm1p(11, 12, 13) ;
-isopentenyl-pyrophosphate: tRNA
isopentenyltransferase, Mod5p(14) ; and ATP(CTP): tRNA-specific
nucleotidyltransferase, Cca1p(15) . Sorting isozymes have
heterogeneous amino-terminal ends which are the result of translation
initiation from more than one in-frame AUG. Selection of the AUG to be
used for translation initiation can depend on whether transcription
initiates upstream or downstream of ATG1 of the ORF (
)(16) . Heterogeneous amino termini can also result
when the translation initiation machinery bypasses an AUG in a
``poor'' context in favor of a downstream
AUG(17, 18, 19) . Translation from the first
ATG provides a mitochondrial targeting signal to promote efficient
import into the organelle while translation from a downstream ATG
eliminates this information and provides enzyme for nuclear and/or
cytoplasmic located tRNA processing.
CCA1 codes for mitochondrial, cytosolic, and nuclear Cca1p which catalyzes the addition of CCA to the 3` termini of tRNA(15, 20, 21) . In yeast, this enzyme is essential because the CCA end is not encoded by either nuclear or mitochondrial tRNA genes (for review, see (2) ). The CCA1 gene has 3 in-frame ATGs at the 5` end of its ORF. An examination of mRNA transcripts from CCA1 demonstrated 5` ends which fall upstream of ATG1 and between ATG1 and ATG2, but not between ATG2 and ATG3. Cca1p synthesis initiates from all three ATGs. This means that translation initiation from ATG3 requires that ribosomes bypass ATG2 to initiate at the downstream ATG3. Thus, an interplay of transcription initiation and translational selection of ATGs leads to the biosynthesis of three isoforms of Cca1p(15, 19) . Site-directed mutagenesis experiments which altered ATG1 demonstrated that protein translated from the first in-frame ATG is necessary for mitochondrial protein synthesis. Mutant genes retaining either or both ATG2 and ATG3 provide nuclear/cytosolic enzyme activities(15, 19) .
The fact that there are three isoforms and three compartments raises the question as to whether each isoform is compartment specific. To address this issue, we investigated the ability of each isozyme to catalyze the addition of CCA to nuclear restricted pre-tRNA by Northern blot analysis. We also examined the cellular distribution of Cca1p by indirect immunofluorescence. Our studies indicate that both Cca1p-II and Cca1p-III are in the nucleus and the cytosol. Cca1p-I is primarily in mitochondria but minor activity from Cca1p-I can also be detected in the nucleus.
We also determined the consequences of altering the cytosolic/nuclear distribution of nucleotidyltransferase activity. By providing Cca1p with a surrogate nuclear localization signal (SV40-NLS-Cca1p) we have substantially reduced the cytosolic pool of Cca1p and concomittantly increased the nuclear pool of this enzyme. At the nonpermissive temperature SV40-NLS-Cca1p is unable to complement a ts cca1-1 allele. This result directly demonstrates an important role for Cca1p in the cytosol of eukaryotes.
We are able to detect by immunoblot analysis Cca1p in extracts of cells containing multiple copies of CCA1. The immunofluorescence experiments reported here were done with cells carrying CCA1 on multicopy vectors, pRS426 (10-20 copies/cell) and pJDB207 (100-200) copies/cell). Fig. 1(A, C, and E) shows the fluorescein isothiocyanate detection of Cca1p immunoreactive complexes. Fig. 1(B, D, and F) shows the 4`,6-diamidino-2-phenylindole (DAPI) staining of nuclear and mitochondrial DNA. A very weak cytosolic signal is detected in cells carrying vector alone and expressing endogenous levels of Cca1p (Fig. 1A). In cells carrying 10-20 copies of CCA1, most Cca1p is in the cytosol with only a little present in nuclei (Fig. 1, C and D). Fig. 1, E and F, shows the subcellular distribution of Cca1p in cells carrying 100-200 copies of CCA1. Again Cca1p is primarily localized to the cytosol. However, we now observe an enrichment of Cca1p in the region surrounding the nucleus.
Figure 1: Subcellular distribution of Cca1p by indirect immunofluorescence. Strain W303-1B was transformed with pRS426 (A and B), CCA1 on pRS426 (C and D), CCA1 on pJDB207 (E and F). An antibody to Cca1p was used to detect the location of Cca1p isozymes. Fluorescein isothiocyanate was used to detect Cca1p immunoreactive complexes (A, C, and E). Nuclear and mitochondrial DNA was stained with DAPI (B, D, and F).
Figure 2: Subcellular distribution of Cca1p isozymes initiating from AUG1, AUG2, and AUG3. Strain W303-1B was transformed with cca1-M1,M3 (A and B), cca1-M1,M2 (C and D), and cca1-M2,M3 (E and F) on pRS426. Antibody to Cca1p was used to detect the location of Cca1p isozymes. Fluorescein isothiocyanate was used to detect Cca1p immunoreactive complexes (A, C, and E). Nuclear and mitochondrial DNA were stained with DAPI (B, D, and F).
Figure 3:
Comparison of nuclear Cca1p isozyme
activity by Northern blot analysis. cca1-1 cells
containing YCp50 (lane 1), cca1-M2,M3 (lane
2), cca1-M1,M3 (lane 3), or cca1-M1,M2 (lane 4) were incubated at the nonpermissive temperature
for 3.5 h. Small RNAs were isolated, separated on a 6% polyacrylamide,
8 M urea gel, and transferred to membrane. The membrane was
probed with an oligonucleotide complementary to the
tRNA intron.
Figure 4: Subcellular distribution of SV40-NLS-Cca1p and SV40-nls-Cca1p by indirect immunofluorescence. Strain W303-1B was transformed with SV40-NLS-CCA1 (A and B) or SV40-nls-CCA1 (C and D) on pRS426. Antibody to Cca1p was used to detect the location of Cca1p isozymes. Fluorescein isothiocyanate was used to detect Cca1p immunoreactive complexes (A and C). Nuclear and mitochondrial DNA were stained with DAPI (B and D).
Figure 5: Growth characteristics of cca1-1 cells transformed with wild-type and mutant CCA1 genes on solid media. Equal amounts of cells were spotted on glucose medium at 25 and 37 °C. Cells were transformed with YCp50, lane 1; CCA1, lane 2; SV40-NLS-CCA1, lane 3; SV40-nls-CCA1, lane 4.
Figure 6: Growth characteristics of cca1-1 cells transformed with wild-type and mutant CCA1 genes in liquid media. Early log phase cells (panel A) and non-log phase cells (panel B) were diluted into synthetic complete uracil minus media and grown at 37 °C. Growth was monitored by optical density measurements at 600 nm.
There are several possible explanations for this. One is that fusion of the SV40 NLS to the amino-terminal end of Cca1p inhibits its enzyme activity. This seems unlikely since SV40-nls-Cca1p, which differs in only one amino acid from SV40-NLS-Cca1p, provides sufficient nucleotidyltransferase activity for wild-type growth. Another is that an increase in nuclear nucleotidyltransferase activity is inhibitory to some other nuclear function essential for normal cell growth. This is unlikely as cells expressing SV40-NLS-Cca1p at the permissive temperature grow fine. The third explanation is that the reduction of the cytosolic pool of Cca1p by nuclear mislocalization is responsible for the growth defect. To address this issue, we examined tRNA biosynthesis in cells carrying each of these constructs on single copy vectors as described below.
Figure 7:
Northern blot analysis of wild-type and
mutant nuclear Cca1p activity. Log phase cca1-1 cells
containing YCp50, lane 1; CCA1, lane 2; SV40-NLS-CCA1, lane 3; and SV40-nls-CCA1, lane 4, were shifted to the nonpermissive temperature and
allowed to grow for 3.5 h. Small RNAs were isolated, separated on a 6%
polyacrylamide, 8 M urea gel, and transferred to membrane. The
membrane was probed with an oligonucleotide complementary to
tRNA intron.
Figure 8:
Northern blot analysis of cytosolic Cca1p
activity in cca1-1 cells at the permissive temperature. Log
phase cells carrying YCp50 (lanes 1 and 5), CCA1 (lanes 2 and 6), SV40-NLS-CCA1 (lanes 3 and 7), or SV40-nls-CCA1 (lanes 4 and 8) on a single copy vector were
incubated at 23 °C for either 4 or 18 h of additional growth. Small
RNAs were isolated, separated on a 6% polyacrylamide, 8 M urea
gel, and transferred to a membrane. The membrane was probed with an
oligonucleotide complementary to tRNA (panel A), tRNA
(panel
B), and tRNA
(panel
C).
Figure 9:
Northern blot analysis of cytosolic Cca1p
activity in cca1-1 cells at the nonpermissive temperature.
Early log phase cells carrying YCp50 (lanes 1 and 5), CCA1 (lanes 2 and 6), SV40-NLS-CCA1 (lanes 3 and 7), or SV40-nls-CCA1 (lanes 4 and 8) on a single copy vector were
incubated at 37 °C for either 4 or 18 h of additional growth. Small
RNAs were isolated, separated on a 6% polyacrylamide, 8 M urea
gel, and transferred to a membrane. The membrane was probed with an
oligonucleotide complementary to tRNA (panel A), tRNA
(panel
B), and tRNA
(panel
C).
Very little end-shortened
tRNA, tRNA
, and
tRNA
accumulates in cca1-1 cells carrying wild-type CCA1 at the nonpermissive
temperature (Fig. 9, lane 2). A trace of
tRNA
and a small fraction of
tRNA
and tRNA
have shortened 3` termini in log phase cca1-1 cells carrying SV40-NLS-Cca1p which produces enzyme largely
confined to the nucleus (Fig. 9, lane 3). In contrast,
end-shortened tRNA
,
tRNA
, and tRNA
do not accumulate in cca1-1 cells carrying
SV40-nls-Cca1p which contains the majority of Cca1p in the cytosol (Fig. 9, lane 4). These results further demonstrate
that Cca1p is required for the repair of cytosolic tRNAs. Although the
ratio of end-shortened to mature tRNA is not as dramatic when nuclear
Cca1p is present (SV40-NLS-CCA1) (Compare Fig. 9, lanes 1 and 3), the slight accumulation of cytosolic
tRNAs, particularly rare tRNAs, lacking 3`-CCA termini could affect
cell growth as seen in Fig. 5and Fig. 6.
In the absence of
nucleotidyltransferase activity, the majority of
tRNA lacks CCA ends (Fig. 9, lanes 1 and 5). Although end-shortened
tRNA
is not detected in exponentially
growing cells producing wild-type Cca1p (Fig. 9, lane
2), a small amount is detected in nonexponentially growing cells (Fig. 9, lane 6). In cells lacking cytosolic Cca1p, the
level of end-shortened tRNA
increases
dramatically during nonexponential growth (Fig. 9, lane
7) compared to exponential growth (Fig. 9, lane
3). Cells carrying SV40-nls-Cca1p do not accumulate 3`
end-shortened tRNA
(Fig. 9, lanes 4 and 8).
End-shortened
tRNA does not accumulate in either log
phase or non-log phase cells carrying SV40-nls-Cca1p (Fig. 9, lanes 4 and 8). Although there is very little
end-shortened tRNA
in exponentially growing
cells carrying wild-type Cca1p (Fig. 9, lane 2) that
amount increases in nonexponentially growing cells (Fig. 9, lane 6). The level of end-shortened tRNA
is greatest in cells lacking cytosolic Cca1p and it is increased
during nonexponential growth (compare Fig. 9, lanes 3 and 7).
ATP(CTP):tRNA nucleotidyltransferase is a sorting isozyme that is localized to the nucleus, mitochondria, and cytosol. Although previous studies demonstrated that mitochondrial enzyme was provided by protein translated from the first in-frame ATG in the CCA1 open reading frame, (19) the source of nuclear and cytosolic activity was not known. Through indirect immunofluorescence studies described here, we have demonstrated that Cca1p-I is highly enriched in mitochondria and that the majority of Cca1p produced from the second and third ATGs in the open reading frame is cytosolic. An examination of pre-tRNAs by Northern blot analysis showed that Cca1p-II and Cca1p-III produced from these ATGs also provide nuclear activity sufficient for normal cell growth and that even Cca1p-I is capable of reaching the nucleus, albeit in amounts too small to support the needs of the cell. Altering the distribution of Cca1p-III such that the majority is located in the nucleus leads to the accumulation of 3` end-shortened tRNAs and causes a growth defect.
Subcellular distribution of nucleotidyltransferase varies among organisms. In rat liver cells, one-third of the nucleotidyltransferase activity is mitochondrial and the remainder is primarily cytosolic (33) . Very little nucleotidyltransferase is found in the nucleus. In Xenopus, however, 30% of nucleotidyltransferase is nuclear (34) . Most of yeast Cca1p is clearly localized to the cytosol. We do not know if the nuclear/cytosolic distribution is a result of strong cytosolic interactions(35) , inefficient nuclear targeting, or a strong nuclear export signal(36, 37, 38) . Since Cca1p does not contain any of the nuclear localization signals described to date (for review, see (39) and 40), we have not examined the strength of its NLS nor have we tried to prevent its delivery to the nucleus. By incorporating the strong SV40 large T antigen NLS into the amino terminus of Cca1p we were able to shift the distribution of Cca1p such that it accumulates in the nucleus. This suggests that a ``weak'' NLS is the determining factor for low levels of nuclear Cca1p.
Cca1p-I is localized primarily to mitochondria. While
the absence of Cca1p-II and Cca1p-III might change the distribution of
Cca1p-I and lead to this accumulation, it is also possible that in
wild-type cells, a small amount of Cca1p-I is normally located to
nuclei and the cytosol. There are two routes that could lead to the
nuclear/cytosolic activity provided by the longest form of Cca1p. First
of all, unlike the mitochondrial form of the enzyme which is processed
upon import (19) Cca1p-I could retain its mitochondrial
targeting signal but not engage with the mitochondrial targeting
machinery. Alternatively, all of the Cca1p-I produced in the cell could
begin the process of import into mitochondria but a portion of it be
released before import is complete as occurs with the enzyme, fumarase.
Fumarase is translated from a single ATG and all of the protein
initiates import into mitochondria and is processed by the
mitochondrial matrix processing enzyme but only a portion continues to
be fully translocated while the majority is released to the
cytosol(41) . Both models for Cca1p are consistent with the
observation that the amino terminus of Cca1p-I does not have the
relatively high hydrophobic moment and the -helical amphipathic
structure characteristic of mitochondrial targeting signals. Indeed,
Mod5p-I, another sorting isozyme with a less than optimal mitochondrial
targeting signal is about evenly divided between mitochondria and the
cytosol(14) . Unfortunately, Mod5p-I is not processed making it
difficult to determine whether all of the protein begins import but
only a portion completes it. In the former scenario, cytosolic
accumulation of Mod5p-I and Cca1p-I could occur if interaction with a
mitochondrial import receptor were compromised. In the latter scenario,
a weak interaction with mitochondrial Hsp70 might lead to retrograde
movement of a portion of the protein(42) . Since Cca1p-I is
processed, we would be able to differentiate between these two models
if we could separate the processed from the unprocessed form and if
there were sufficient Cca1p-I in the cell to detect. Unfortunately,
Cca1p does not fulfill either of these criteria.
Our studies have also allowed us to address the functional role of the cytosolic pool of nucleotidyltransferase. Cca1p-II and Cca1p-III provide the bulk of nuclear/cytosolic enzyme activity with the largest fraction of Cca1p-II and Cca1p-III remaining in the cytosol. Unlike nuclear and mitochondrial located Cca1p, cytosolic Cca1p does not play a role in tRNA biosynthesis. Instead, cytosolic Cca1p catalyzes the repair of 3` end-shortened tRNA.
Yeast cells that lack cytosolic nucleotidyltransferase exhibit a growth phenotype. This phenotype is relatively mild in early log phase cells with only a 30% increase in doubling time. However, non-log phase cells are unable to properly regain log phase growth when diluted into synthetic complete minus uracil media (Fig. 6). An increase in the cytosolic pool of 3` end-shortened tRNA accompanies this growth defect in both phases of cell growth, suggesting that this accumulation of end-shortened tRNA is responsible for the growth phenotype.
Cytosolic Cca1p is
functionally analogous to E. coli nucleotidyltransferase; for
the only known function of E. coli nucleotidyltransferase is
in repair, since E. coli tRNAs are transcribed with CCA
ends(43) . In E. coli, aminoacylation or charging
protects the 3` termini of tRNA from ribonucleases(43) .
However, the efficiency of charging varies among tRNAs. Consequently in E. coli cells that lack nucleotidyltransferase activity, 10%
of tRNAs have 90% of the end-shortened termini(43) . In yeast,
as in E. coli, tRNAs differ in their susceptibility to
ribonuclease removal of their 3` CCA end. In cells lacking
nucleotidyltransferase activity, almost all tRNA has lost its 3` terminal CCA sequence; whereas much of
tRNA
retains its CCA end. Although a large
fraction of tRNA
also contains a CCA end, a
sizeable portion of tRNA
does not (Fig. 9).
More end-shortened tRNAs accumulate in non-log phase cells than in log phase cells, at least for the tRNAs we examined (see Fig. 9, lanes 3 and 7). One explanation for the difference in accumulation of end-shortened tRNA is that more tRNA is synthesized in log phase cells allowing them to maintain a pool of mature cytosolic tRNA which serves to lessen the growth phenotype. This idea is supported by the observation that in cells that lack all nucleotidyltransferase activity, a much greater proportion of the tRNAs examined is end-shortened (Fig. 9, lanes 1 and 5). Another explanation is that, as in E. coli(43) , there is an increase in ribonuclease catalyzed removal of the 3` CCA end during non-log phase growth. Thus, it is possible that changes in de novo synthesis of tRNA and ribonuclease activity both contribute to the differences observed in the cytosolic pool of end-shortened tRNAs in log phase and non-log phase cells.
The accumulation of a pool of end-shortened uncharged tRNA causes a growth defect in yeast. One possible model to explain this is that uncharged tRNA competes with charged tRNA for the A site on the ribosome. This competition might result in ribosomal stalling and induction of a ``stringent response'' similar to that observed in E. coli. The stringent response is characterized by a decrease in tRNA synthesis, rRNA synthesis, rRNA transcription, protein synthesis, and an increase in amino acid biosynthesis(44) . A second model is that accumulation of uncharged tRNA induces a response similar to that induced by amino acid starvation. In this case, uncharged tRNA levels leads to the induction of the GCN2 catalyzed phosphorylation of translation initiation factor, eIF-2e. Phosphorylation of eIF-2, leads to down-regulation of protein synthesis and cell division as well as up-regulation of transcription factor GCN4 which activates increased expression of amino acid biosynthetic genes(45, 46) .