- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Email this article to a friend
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Tolerico, L. H.
- Articles by Hopper, A. K.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Tolerico, L. H.
- Articles by Hopper, A. K.
Saccharomyces cerevisiae Mod5p-II Contains Sequences Antagonistic for Nuclear and Cytosolic Locations
Leslie H. Tolerico1,2,a, Ann L. Benko1,b, John P. Arisc, David R. Stanfordb, Nancy C. Martind, and Anita K. Hoppera,ba Program in Cell and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033,
b Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033,
c Department of Anatomy and Cell Biology, University of Florida Health Science Center, Gainesville, Florida 32610
d Department of Biochemistry, University of Louisville Medical School, Louisville, Kentucky 40292
Corresponding author: Anita K. Hopper, Department of Biochemistry and Molecular Biology H171, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033., ahopper{at}psu.edu (E-mail)
Communicating editor: F. WINSTON
 | ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
MOD5 encodes a tRNA modification activity located in three subcellular compartments. Alternative translation initiation generates Mod5p-I, located in the mitochondria and the cytosol, and Mod5p-II, located in the cytosol and nucleus. Here we study the nucleus/cytosol distribution of overexpressed Mod5p-II. Nuclear Mod5p-II appears concentrated in the nucleolus, perhaps indicating that the nuclear pool may have a different biological role than the cytoplasmic and mitochondrial pools. Mod5p contains three motifs resembling bipartite-like nuclear localization sequences (NLSs), but only one is sufficient to locate a passenger protein to the nucleus. Mutations of basic residues of this motif cumulatively contribute to a cytosolic location for the fusion proteins. These alterations also cause decreased nuclear pools of endogenous Mod5p-II. Depletion of nuclear Mod5p-II does not affect tRNATyr function. Despite the NLS, most Mod5p is cytosolic. We assessed whether Mod5p sequences cause a karyophilic reporter to be located in the cytosol. By this assay, Mod5p may contain more than one region that functions as cytoplasmic retention and/or nuclear export sequences. Thus, distribution of Mod5p results from the presence/absence of mitochondrial targeting information and sequences antagonistic for nuclear and cytosolic locations. Mod5p is highly conserved; sequences responsible for subcellular distribution appear to reside in "accessory" motifs missing from prokaryotic counterparts.
SORTING isozymes are located in multiple subcellular locations but they are encoded by a single gene (
Among genes encoding sorting isozymes, the Saccharomyces cerevisiae MOD5 and CCA1 genes are unusual in encoding isozymes located in three, rather than two, locations: mitochondria, nuclei, and the cytosol (
MOD5 encodes two isozymes, Mod5p-I and Mod5p-II, initiating at codons 1 and 12 of the open reading frame (ORF), respectively (
Several different types of cis-acting sequences can deliver karyophilic proteins to the nucleus. These include the SV40 large T antigen nuclear localization sequence (NLS) PKKKRKV (
Many eukaryotic proteins are found in both the nucleus and the cytosol. Several mechanisms regulate the protein distribution of these two subcellular compartments (for a review, see
| MATERIALS AND METHODS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Strains, media, and transformation:
The Saccharomyces cerevisiae strain MT-8 [MAT
ura3-1 leu2-3,112 ade2-1 trp1 lys1-1 lys2-1 SUP7 can1-100 mod5::TRP1 (
Escherichia coli RR1 and DH5 were used for propagation of recombinant DNA constructs. RZ1032 was used for propagation of M13 phage, and RR1 and JM109 were recipients for mutagenesis reactions. E. coli were maintained on yeast extract tryptone (YT) media or YT media containing the appropriate antibiotic to select for plasmid expression. E. coli were transformed using the calcium chloride procedure (
Oligonucleotide generation and PCR amplification:
Oligonucleotides were prepared by the Pennsylvania State University College of Medicine Macromolecular Core Facility. Table 1 lists the sequences of the oligonucleotides that were used as primers. Table 2 lists the PCR products used in this study and the primer pairs and templates used in their synthesis.
|
|
Oligonucleotide-directed mutagenesis:
Four mutant alleles of MOD5 were generated essentially as described by
Plasmid construction:
Sequences of MOD5 PCR products and mutated alleles were determined by the dideoxy-chain termination method (
MOD5 PCR products were initially ligated into pGEM-T (Promega, Madison, WI), except for MOD5(12–234) and MOD5(230–403), which were ligated into pBlueScriptSK(-) (Stratagene, La Jolla, CA). These intermediate constructs were then digested with BamHI and the subcloned PCR fragments were released and isolated. DNA fragments MOD5(102–203), MOD5(375–427), mod5-KRKNN, mod5-KRNNN, and mod5-NNNNN were each ligated into the BamHI site of the vector pFB1-7a (
|
MOD5 mutant alleles were released from pBlueScriptSK(+) by digestion with BstYI and were cloned into the BamHI site of YCf50 (Table 3). All constructs were subsequently cloned into the unique BamHI site of pJDB207 (Table 3).
The plasmids used for the green fluorescent protein (GFP) studies were constructed as follows. The open reading frame for Aequorea victoria GFP from pSEY18/GAL1-10/GFP was PCR-amplified using primers MOD5GFP5' and MOD5GFP3', which contain NotI sites. The resulting product, GFP/NotI, was subcloned into the pGEM-T vector (Promega). GFP was isolated from the vector through a NotI restriction digest and inserted into the NotI site of pBlueScribemod5-M2KR6.Not (
Sequence analysis:
The S. cerevisiae Mod5p (GenBank accession no. 2507067) sequence was BLASTed (
Characterization of monoclonal antibody 32D6:
The monoclonal antibody 32D6 was generated against a nucleolusenriched fraction from yeast nuclei consisting of nucleoli with attached nuclear envelopes that contain nuclear pore complexes (
Indirect immunofluorescence and microscopic imaging:
Indirect immunofluorescence experiments were carried out essentially as described by
A 1:5 dilution of affinity-purified rabbit-anti-Mod5p antibody (
For double staining procedures, cells expressing wild-type MOD5 on pJDB207 were fixed for 30 min and were stained with a 1:5 dilution of anti-Mod5p antibody in addition to either 32D6 or anti-Nop1p antibodies. Antibody 32D6 was used in a 1:20,000 dilution to detect nuclear pore complex proteins. The anti-Nop1p antibody (
To observe moderately overexpressed GFP fusion protein and DNA in the same cells, MT-8 cells containing either pRS426mod5-M1-GFP or pRS426mod5-M2-GFP were grown overnight in selective media containing 10 ng/ml 4',6-diamidino-2-phenylindole (DAPI). To visualize GFP fusion protein encoded by pRS416mod5-M1-GFP in MT-8, cells were grown overnight on plates at 23° and then suspended in phosphate-buffered saline prior to microscopy.
Fluorescence images were obtained by using either a Nikon Microphot-FX or a Nikon Optiphot-2 microscope equipped with a SenSys CCD camera (Photometrics Ltd., Tucson, AZ) and a Nikon 35-mm camera. Image processing for Figure 1, Figure 6, Figure 7, and Figure 8 was done using IPLab Spectrum software (Signal Analytics Corp., Vienna, VA); image processing for Figure 3 and Figure 9 employed QED software (Pittsburgh). Images for Figure 5 were obtained using a 35-mm camera and TMAX ASA 400 film (Eastman Kodak Co., Rochester, NY).
|
|
|
|
|
|
|
|
|
ß-Galactosidase assay:
ß-Galactosidase activity was measured with a modified version of the method of
| RESULTS |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Nucleus-located Mod5p-II appears to be concentrated in the nucleolus:
Previously, employing cell fractionation procedures using yeast cells containing endogenous levels of Mod5p or immunofluorescence procedures using yeast cells containing overexpressed Mod5p, we reported that Mod5p-II was located in both the cytosol and nucleus of yeast cells (
The specificity of the nuclear pore complex antibody, mAb 32D6, was demonstrated as follows: first, it recognizes a nuclear pore complex antigen by immunofluorescent localization; second, in a 
gt11 screen 32D6 identified a single clone with ~66% of the C-terminal portion of NSP1; third, we show here that proteins from strain RS453, which expresses a short form of Nsp1p, and from the wild-type strain BJ5465 migrate on SDS gels as expected (Figure 2). NSP1 in RS453 contains an internal deletion that removes the coding sequences for six nonessential FXFG repeats, encoding a protein migrating at ~85 kD on SDS gels (E. HURT, personal communication). Wild-type Nsp1p migrates on SDS gels at ~100 kD (
As endogenous Mod5p cannot be detected by immunofluorescence (
Since a portion of the Mod5p-II pool is inside the nuclear border, but apparently adjacent to DNA, we hypothesized that it may be located in the nucleolus. To determine whether Mod5p staining coincides with the nucleolus, yeast cells harboring pJDB207 (Figure 1B, A–C), pJDBmod5-M1 (Figure 1B, D–F), or pJDBMOD5 (Figure 1B, G–I) were double stained with anti-Mod5p antibody and anti-Nop1p, the latter an established nucleolar protein (mAb A66;
As overexpression of Mod5p-II could lead to accumulation in an inappropriate subcellular compartment, we attempted to view Mod5p-II in cells expressing lower levels. Plasmids pRS416mod5-M1-GFP, pRS426mod5-M1-GFP, and pRS426mod5-M2-GFP encode Mod5p-GFP fusion proteins that complement the mod5::TRP1 disruption allele (data not shown). Fusion proteins encoded by episomal plasmid pRS426mod5-M1 are located in the cytosol and the nucleus. Often two putative nuclear bodies are visualized (Figure 3B), only one of which overlaps with DAPI staining of DNA (compare Figure 3B and B'). The pool of Mod5p that appears nuclear but does not overlap with DNA is likely nucleolar. However, the identity of the nucleolus cannot be confirmed because it is not possible to double stain living cells with two different fluorochromes.
The centromere-containing vector pRS416mod5-M1-GFP construct generated very faint signals (Figure 3A) that could be viewed only when amplified with a CCD camera and could not be counterstained due to UV signal overlapping in the GFP channel. However, the results indicate that in moderate-copy and in low-copy vectors, Mod5p-II is nucleoplasmic and nucleolar in living cells, as it is when expressed from high-copy vectors in fixed cells. As previously reported for fixed cells (
The Mod5p-II carboxyl terminus contains a bipartite-like NLS sequence sufficient to deliver a passenger protein to the nucleus:
Mod5p-II possesses three sequences that resemble the bipartite NLS motif, which consists of two basic clusters separated by a 10-amino-acid spacer. The first two sequences, 122KRVDTKSSERKLTRK136 and 170RRVQRMLEIYYKTGKK185, are located in the N-terminal half of Mod5p. The third sequence, 408KRNTRQADFEKWKINKK424, is located near the C terminus of the protein (Figure 4). To determine whether the Mod5p sequences that resemble the bipartite NLS motif have NLS activity, we tested whether regions of Mod5p containing them were sufficient to target a passenger protein to the nucleus. pFB1-7a encodes a cytoplasmic protein with the first 14 amino acids of histone H2B fused in-frame to ß-galactosidase (
ß-Galactosidase in cells harboring pFB1-7a (Figure 5A and Figure B) was distributed throughout the cells, with no nuclear accumulation. Cells expressing pFB1-67a (Figure 5C and Figure D) displayed nearly exclusive nuclear staining. Expression of pFB1-7aMOD5(102–203) (Figure 5E and Figure F), encoding the two N-terminal putative Mod5p NLSs, resulted in staining indistinguishable from that obtained for plasmid pFB1-7a. In contrast, cells expressing pFB1-7aMOD5(375–427) (Figure 5G and Figure H), which encodes the C-terminal putative Mod5p NLS, displayed nuclear staining indistinguishable from the results obtained for pFB1-67a. Thus, pFB1-7aMOD5(375–427) encodes a protein with NLS activity.
To determine whether the NLS activity of the protein encoded by pFB1-7aMOD5(375–427) maps to the sequence that resembles the bipartite NLS motif, we altered the candidate Mod5p NLS consensus sequence and determined the locations of the resulting fusion proteins. Previous studies have shown that substitution of asparagines for basic amino acids of the bipartite motif destroys NLS activity and that the two halves of the NLS are interdependent (
|
The carboxyl terminal bipartite-like NLS is necessary for the nuclear location of Mod5p-II:
The above studies show that Mod5p amino acids 408–424 have NLS activity. However, they do not show that this motif is important for the nuclear location of authentic Mod5p. Nor do they eliminate the possibility that the two other Mod5p sequences (at 122–136 and 170–185) that resemble the bipartite NLS consensus but did not appear to have NLS activity by the passenger protein assay do have NLS activity in endogenous Mod5p. It is also possible that sequences other than those we tested could be important for the nuclear pool of Mod5p. Therefore, we tested whether the motif located at 408–424 was necessary for the nucleus location of Mod5p-II by altering the same basic amino acids of this motif in Mod5p-II. Mutations of the three lysines of Mod5p at 420, 423, and 424 to asparagines generated a mutated sequence Mod5p-KRNNN and mutations of lysines at 408, 409, 420, 423, and 424 generated Mod5p-NNNNN. The altered sequences were subcloned to create plasmids that have the first ATG destroyed and, therefore, encode only the Mod5p-II variant.
Each construct was transferred to a centromere-containing vector to assess Mod5p activity when the protein was expressed in approximately endogenous quantities (Table 3 and Table 5). Since i6A modification affects the efficiency of suppression by the nuclear-encoded suppressor tRNA SUP7, inactivation of MOD5 can be assessed by the levels of nonsense suppression of lys1-1, lys2-1, and ade2-1. Cells with the ade2-1 allele accumulate a red pigment, generating red colonies on rich media, and cells with lys1-1, lys2-1, and ade2-1 are unable to grow on media lacking lysine or adenine. Cells expressing vector alone (YCf50) produce no Mod5p and are unable to suppress the nonsense alleles. Conversely, cells expressing YCfMOD5 produce roughly endogenous levels of cytoplasmic Mod5p-I and Mod5p-II and completely suppress lys1-1, lys2-1, and ade2-1. Cells expressing YCfmod5-M1, which produces only Mod5p-II, have suppression indistinguishable from wild type. The suppression phenotypes of cells harboring the mod5-KRNNN and mod5-NNNNN mutant alleles were indistinguishable from the parental counterparts, demonstrating that proteins encoded by these constructs are fully functional.
|
Extracts were obtained from MT-8 cells expressing various forms of Mod5p-II in the high-copy vector, pJDB207 (Table 3). The quantities and mobilities of proteins from each strain were detected by Western blot analysis using affinity-purified Mod5p antibody. The blot was counterstained with an actin-specific antibody to confirm equal loading of protein. By this assay each construct produced roughly equivalent levels of protein of the appropriate electrophoretic mobility (data not shown).
To determine the effects of mutating the putative NLS on the nuclear localization of Mod5p-II, the mutant constructs were expressed from the pJDB207 multicopy plasmid and mutant proteins were localized by indirect immunofluorescence. Cells expressing the pJDB207 vector alone revealed no Mod5p-specific staining (Figure 7A and Figure B). Cells harboring pJDBmod5-M1 (Figure 7C and Figure D), which produces only Mod5p-II, as expected, had both cytosol and nuclear (nucleolar) Mod5p. pJDBmod5-M1-KRNNN (Figure 7E and Figure F) resulted in only a small increase in the level of cytoplasmic Mod5p and a concomitant decrease in nuclear accumulation of the mutant isozyme, whereas the protein encoded by pJDB207mod5-M1-NNNNN (Figure 7G and Figure H) appeared to be located primarily in the cytosol. The data show that the C-terminal NLS-like sequence of Mod5p is important for nuclear accumulation of the overexpressed Mod5p-II isozyme and that the motif at 408–424 is the only NLS in the MOD5 sequence.
Cytoplasmic retention/nuclear export sequence(s) may regulate the nucleus/cytosol distribution of Mod5pII:
Mod5p-II resides in both the nucleus and cytoplasm. Although the putative Mod5p NLS appears to be a "strong" targeting sequence, as it can quantitatively deliver a passenger protein to nuclei, it does not completely localize Mod5p-II to the nucleus. Several possibilities could account for the cytoplasmic pool. One is that the protein may fold in such a manner that the NLS is not optimally exposed or easily accessible to the import machinery, thereby decreasing its efficiency in nuclear import. To determine whether NLS occlusion might explain cytosolic Mod5p-II, we inserted DNA encoding the SV40 large T antigen NLS into plasmids harboring the mod5-M1 allele just downstream of the second ATG of the MOD5 ORF. The amino terminus was chosen since presumably it is an exposed area of the protein that is able to present the mitochondrial targeting information to the import machinery. The SV40 large T antigen NLS has been shown to function efficiently in yeast (
To determine the effect of the addition of the SV40 large T antigen NLS on nuclear import of Mod5p-II, the mod5-M1-SV40 allele was expressed in multicopy in the yeast MT-8 and the location of the resulting protein was analyzed by indirect immunofluorescence (Figure 8, compare A–D with E and F). While there may be a slight increase in the nuclear accumulation of Mod5p-II-SV40, this isozyme was not efficiently delivered to the nucleus. The inability of Mod5p-II-SV40 to efficiently locate to the nucleus indicates that the cytoplasmic pool of this isozyme is unlikely to be due to masking of the Mod5p NLS.
We considered 3' mRNA end heterogeneity (
Cells expressing pJDBmod5-M1-KRK did not show a decrease in nuclear staining (Figure 8, compare C and D with G and H). These results are consistent with our other studies (Figure 6) showing that mutations of the downstream basic amino acids of the Mod5p NLS have little effect on the ability of the NLS to function in the delivery of a passenger protein to the nucleus. The data indicate that 3' mRNA heterogeneity is an unlikely mechanism to account for the cytosolic pool of Mod5p-II.
As NLS masking and 3' mRNA heterogeneity do not seem to regulate the nucleus/cytosol distribution of Mod5p-II, we considered the possibility that Mod5p-II could possess sequences that act either to retain a portion of the pool of this protein in the cytosol (CRD or CRS) or, alternatively, that act to redistribute nucleus-located Mod5p-II to the cytosol (NES). Even though Mod5p-II does not possess motifs identical to characterized CRD, CRS, or NES sequences, Mod5p-II may possess cis-regions of different sequence that fulfill similar functions. To identify such putative sequences we employed an assay to test for their function. Plasmid pFB1-67a contains the codons for the first 67 amino acids of histone H2B that include the histone NLS fused in-frame to ß-galactosidase. The resulting H2B/ß-galactosidase fusion protein is located in the nucleus of yeast cells (
Initially we assessed the location of fusion proteins possessing nearly the entire Mod5p-II. The fusion protein encoded by pFB1-67aMOD5(12–427) is located primarily in the cytosol (Figure 9B and Figure 10). Although the protein could be located in the cytosol because it is misfolded and the NLS is masked, this is not likely as the fusion protein has similar activity to the protein encoded by pFB1-67a (Table 4). To map the sequences responsible for the cytosolic location of the fusion protein we generated two plasmids each encoding about one-half of Mod5p-II. pFB1-67aMOD5(12–234) contains MOD5 codons 12–234 whereas pFB1-67aMOD5(230–403) contains MOD5 codons 230–403. The plasmids were transferred to strain MT-8, and the activities and locations of H2B/Mod5p/ß-galactosidase proteins were determined. Surprisingly, both fusion proteins located primarily in the cytosol (Figure 9C and Figure D and Figure 10). Again, the cytosolic location did not appear to be an artifact of grossly misfolded proteins as the fusion proteins had about the same level of activities as the starting H2B/ß-galactosidase protein (Table 4). We considered the possibility that Mod5p-II contains more than one cis-acting sequence regulating its nucleus/cytosolic distribution. To further map the putative regulatory sequences, the sequences in pFB1-67MOD5(12–234) and pFB1-67aMOD5(230–403) were each divided into two halves generating plasmids that individually contained Mod5p amino acids 12–109, 102–234, 230–326, and 324–403, respectively (Figure 9, E–H and 10). Each encoded the appropriate ß-galactosidase activity (Table 4). Fusion proteins with Mod5p amino acids 12–109 or 324–403 were located primarily in the nucleus (Figure 9E and Figure H) whereas proteins with Mod5p amino acids 102–234 or 230–326 were located in the cytosol and nucleus (Figure 9F and Figure G). However, there was heterogeneity among cells with respect to location of these latter two fusions. Perhaps the entire region causing a cytosolic pool is not included in either of them.
|
Mod5p amino acid regions 197–206 and 225–236 are leucine-rich and resemble the NES consensus motif. To investigate the possibility that either/both of these sequences might function in nuclear export, additional fine mapping in this vicinity of Mod5p was performed. The H2B/Mod5p/ß-galactosidase proteins encoded by pFB1-67aMOD5(170–243), pFB-67aMOD5(206–243), pFB1-67aMOD5(206–276), pFB1-67aMOD5(230–276), and pFB1-67aMOD5(275–326) were found to have enzymatic activities similar to H2B/ß-galactosidase alone (Table 4). Proteins with Mod5p amino acids 206–276 were predominantly cytosolic (Figure 10). Removal of sequence from either the carboxyl terminus (in proteins with amino acids 170–243 and 206–243) or from the amino terminus (in proteins with amino acids 230–276) of this Mod5p region resulted in increased nuclear pools or heterogeneous distribution. Fusion proteins containing amino acids 275–326 were also located in both the nucleus and cytosol. Even though our studies indicate that Mod5p-II possesses sequences in the middle of the protein that are responsible for the cytosolic pool, we have been unable to define them precisely because they appear to be redundant, long, and complicated. (However, see below.)
Conservation of regions implicated for Mod5p subcellular distribution:
Genome sequencing has uncovered putative MOD5 homologs from many organisms. For eukaryotes, there are sequences that would encode the homologs for C. elegans and S. pombe and there are partial sequences for the putative human, Drosophila melanogaster, and Plasmodium homologs. For eubacteria, 33 sequences for the putative MOD5 homologs (mia in E. coli) are in the databases. None of the sequenced archaeal genomes contains a MOD5/miaA gene. All of the eukaryotic and eubacteria sequences share striking conservation. However, the eukaryotic sequences have additions. The S. cerevisiae and C. elegans sequences possess short amino-terminal additions and two in-frame AUGs at codons 1 and 12 or 15, respectively (Figure 11). The GenBank entry for the C. elegans MOD5 gene (U13642) begins the coding sequence at the second AUG; however, an in-frame upstream AUG is present and the sequence can be translated as: MIFRKFLNFLKPYKM... In yeast, the N-terminal 11 Mod5p amino acids are a necessary part of the mitochondrial targeting sequence (MTS;
|
Yeast and C. elegans MOD5 genes each encode an ~100-amino-acid carboxyl motif (yeast Mod5p amino acids 303–344 and 373–428) not found in prokaryotes. Here, we show that the yeast NLS is located near the end of this C-terminal extension (amino acids 408–424). We did not find a perfect match to either the SV40 large T antigen or the bipartite-like NLS consensus by inspection of the C. elegans carboxyl-terminal extension. However, in a location close to the Mod5p NLS the C. elegans has an imperfect match to the bipartite NLS consensus: KHIDGKKHKHHAKQKK (Figure 11, underlined sequences). The similarities lead us to predict a nuclear pool of the C. elegans Mod5p.
Yeast, C. elegans, and Plasmodium Mod5p sequences each possess two short internal stretches of amino acids that are absent or different from the bacterial counterparts (yeast Mod5p amino acids 238–257 and 273–277; Figure 11). These sequences overlap with the location of one of the putative export/retention domains we identified (amino acids ~230–326) perhaps providing auxiliary evidence that they have a role other than catalysis. Future experiments guided by the information regarding eukaryotic additions may help delineate more precisely those sequences involved in nucleus/cytosol distribution. In sum, it appears that the eukaryotic S. cerevisiae and C. elegans Mod5p have acquired "accessory" sequences that are important to the cellular distribution of this highly conserved family of proteins. Use of two in-frame AUGs allows for the synthesis of proteins containing or lacking part of the accessory information.
| DISCUSSION |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Mod5p-II cis-acting sequences important for the nuclear pool:
Three lines of evidence support the conclusion that the carboxyl terminal motif located between amino acids 408 and 424 is the Mod5p-II NLS. First, this sequence conforms exactly to the bipartite NLS consensus sequence [KR (X10) 3 of 5 K or R]. Second, a region of Mod5p-II containing this sequence is sufficient to target a passenger protein to the nucleus and mutations of the consensus amino acids affect the efficiency of nuclear accumulation of the passenger protein. Third, this motif is necessary for the nuclear pool of Mod5p.
Two other Mod5p-II regions resemble the bipartite NLS consensus. Amino acids 122–136 and 170–185 each contain the requisite number of basic amino acids, but the two basic regions are separated by 8 and 9 amino acids, rather than the conserved 10-amino-acid spacer (
Although the Mod5p-II motif located between amino acids 408 and 424 appears to be a bonafide NLS, it differs in some respects from other bipartite NLS (
Remarkably, alteration of all consensus basic amino acids to asparagines does not completely destroy NLS activity for ß-galactosidase. It is possible that, as others have recently shown (for a review, see
Regulation of the nucleus/cytosol pools of Mod5p-II:
Mod5p-II possesses a bipartite NLS that promotes efficient nuclear accumulation of ß-galactosidase. However, subcellular fractionation of cells expressing endogenous MOD5 or Mod5p from multicopy vectors (
The presence of nuclear export or cytosol retention sequences in Mod5p-II could generate the cytosolic pool. By inspection, Mod5p does not contain any closely matched sequences to the previously characterized NES, CRS, or CRD motifs (
Role of the nuclear pool of Mod5p-II and nucleolar location:
The initial discovery of nuclear Mod5p-II was surprising in view of the studies of the processing of yeast pre-tRNATyr in Xenopus oocytes that showed i6A moiety addition to tRNA only after intron removal, which occurs just prior to nuclear export (for a review, see
If the nuclear pool is not essential for tRNA modification, why should there be a nucleus-located pool of this enzyme? Here we show that nuclear Mod5p-II appears to be concentrated in the nucleolus. Overexpression is necessary to view the subnuclear location of Mod5p-II, so it is possible that the nucleolar location is an artifact of overproduction. However, as the location of other tRNA processing activities does not change upon overproduction (for example, compare
| FOOTNOTES |
|---|
1 These authors contributed equally to this work. 
2 Present address: Department of Neuroscience, Brown University, Providence, RI 02912.
| ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the American Cancer Society and the National Science Foundation to A.K.H. and N.C.M. and from the National Institutes of Health to J.P.A. We thank Dr. E. Hurt for yeast strains and unpublished information, Dr. D. Engelke for information prior to publication, and Dr. J. Warner for suggestions concerning simultaneous viewing of GFP and DAPI. We also thank Dr. G. Vaduva and L. Palmer for their assistance in the creation of the Mod5p-GFP constructs.
Manuscript received May 22, 1998; Accepted for publication September 24, 1998.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHAFFER, J. ZHANG, and Z. ZHANG et al., 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402
ARIS, J. P. and G. BLOBEL, 1988 Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol. 107:17-31
ATTARDI, G. and G. SCHATZ, 1988 Biogenesis of mitochondria. Annu. Rev. Cell Biol. 4:289-333.
BEGGS, J. D., 1991 Multiplecopy yeast plasmid vectors, pp. 383–390 in Molecular Genetics in Yeast, Alfred Benzon Symposium, Vol. 16, edited by C. VON WETTSTEIN, J. FRIIS, M. KIELLAND-BRANDT and A. STENDERUP. Munksgaard, Copenhagen.
BENTON, B. M., W.-K. ENG, J. J. DUNN, F. W. STUDIER, and R. STERNGLANZ et al., 1990 Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. Mol. Cell. Biol. 10:353-360
BERTRAND, E., F. HOUSER-SCOTT, A. KENDALL, R. H. SINGER, and D. R. ENGELKE, 1998 Nucleolar localization of early tRNA processing. Genes Dev. 12:2463-2468
BOGUTA, M., L. A. HUNTER, W.-C. SHEN, E. C. GILLMAN, and N. C. MARTIN et al., 1994 Subcellular localization of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondria and nuclear isoforms commingle in the cytosol. Mol. Cell. Biol. 14:2298-2306
CHAMBERLAIN, J. R., E. PAGÁN-RAMOS, D. W. KINDELBERGER, and D. R. ENGELKE, 1996 An RNase P RNA subunit mutation affects ribosomal RNA processing. Nucleic Acids Res. 24:3158-3166
CHEN, D.-C., B.-C. YANG, and T.-T. KUO, 1992 One-step transformation of yeast in stationary phase. Curr. Genet. 21:83-84[Medline].
CHEN, S. P., J. S. BROCKENBROUGH, J. E. DOVE, and J. P. ARIS, 1997 Homocitrate synthase is located in the nucleus in the yeast Saccharomyces cerevisiae.. J. Biol. Chem. 272:10839-10846
CHRISTIANSON, T. W., R. S. SIKORSKI, M. DANTE, and P. HIETER, 1992 Multifunctional yeast high-copy number shuttle vectors. Gene 110:119-122[Medline].
CLARK, M. W. and J. ABELSON, 1987 The subnuclear localization of tRNA ligase in yeast. J. Cell Biol. 105:1515-1526
DANPURE, C. J., 1995 How can the products of a single gene be localized to more than one intracellular compartment? Trends Cell Biol. 5:230-238[Medline].
DE BEUS, E., J. S. BROCKENBROUGH, B. HONG, and J. P. ARIS, 1994 Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker. J. Cell Biol. 127:1799-1813
DIHANICH, M. E., D. NAJARIAN, R. CLARK, E. C. GILLMAN, and N. C. MARTIN et al., 1987 Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.. Mol. Cell. Biol. 7:177-184
DINGWALL, C. and R. A. LASKEY, 1991 Nuclear targeting sequences—a consensus? Trends Biochem. Sci. 16:478-481[Medline].
DOVE, J. E., J. S. BROCKENBROUGH and J. P. ARIS, 1998 Isolation of nuclei and nucleoli from the yeast Saccharomyces cerevisiae, pp. 33–46 in Nuclear Structure and Function, Vol. 53, edited by M. BERRIOS. Academic Press, San Diego.
FISCHER, U., J. HUBER, W. C. BOELENS, I. W. MATTAJ, and R. LÜHRMANN, 1995 The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475-483[Medline].
GERACE, L., 1995 Nuclear export signals and the fast track to the cytoplasm. Cell 82:341-344[Medline].
GIETZ, D., A. ST. JEAN, R. A. WOODS, and R. H. SCHIESTL, 1992 Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20:1425
GILLMAN, E. C., L. B. SLUSHER, N. C. MARTIN, and A. K. HOPPER, 1991 MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA. Mol. Cell. Biol. 11:2382-2390
HANAHAN, D., J. JESSEE, and F. R. BLOOM, 1991 Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 204:63-113[Medline].
HENRIQUEZ, R., G. BLOBEL, and J. P. ARIS, 1990 Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen. J. Biol. Chem. 265:2209-2215
HIGGINS, D. G., J. D. THOMPSON, and T. J. GIBSON, 1996 Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266:383-402[Medline].
HOPPER, A. K., and N. C. MARTIN, 1992 Processing of yeast cytoplasmic and mitochondrial precursors to yeast cytoplasmic transfer RNAs, pp. 99–141 in The Molecular Biology of the Yeast Saccharomyces: Gene Expression, Vol. II, edited by E. W. JONES, J. R. PRINGLE and J. R. BROACH. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
HOPPER, A. K., A. H. FURUKAWA, H. D. PHAM, and N. C. MARTIN, 1982 Defects in modification of cytoplasmic and mitochondrial transfer RNAs are caused by single nuclear mutations. Cell 28:543-550[Medline].
HOPPER, A. K., H. M. TRAGLIA, and R. W. DUNST, 1990 The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J. Cell Biol. 111:309-321
HURT, E. C., 1988 A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Saccharomyces cerevisiae.. EMBO J. 7:4323-4334[Medline].
JANS, D. A. and S. HÜBNER, 1996 Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol. Rev. 76:651-685
KALDERON, D., W. D. RICHARDSON, A. F. MARKHAM, and A. E. SMITH, 1984 Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature 311:33-38[Medline].
KILMARTIN, J. V. and A. E. M. ADAMS, 1984 Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces.. J. Cell Biol. 98:922-933
KUNKEL, T. A., J. D. ROBERTS, and R. A. ZAKOUR, 1987 Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382[Medline].
LEE, B., A. G. MATERA, D. C. WARD, and J. CRAFT, 1996 Association of RNase mitochondrial RNA processing enzyme with ribonuclease P in higher ordered structures in the nucleolus: a possible coordinate role in ribosome biogenesis. Proc. Natl. Acad. Sci. USA 93:11471-11476
LI, J.-M., A. K. HOPPER, and N. C. MARTIN, 1989 N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae.. J. Cell Biol. 109:1411-1419
MAKKERH, J. P. S., C. DINGWALL, and R. A. LASKEY, 1996 Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Curr. Biol. 6:1025-1027[Medline].
MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
MARTIN, N. C. and A. K. HOPPER, 1994 How single genes provide tRNA processing enzymes to mitochondria, nuclei and the cytosol. Biochimie 76:1161-1167[Medline].
MICHAEL, W. M., M. CHOI, and G. DREYFUSS, 1995 A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell 83:415-422[Medline].
MICHAEL, W. M., P. S. EDER, and G. DREYFUSS, 1997 The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. EMBO J. 16:3587-3589[Medline].
MORELAND, R. B., G. L. LANGEVIN, R. H. SINGER, R. L. GARCEA, and L. M. HEREFORD, 1987 Amino acid sequences that determine the nuclear localization of yeast Histone 2B. Mol. Cell. Biol. 7:4048-4057
MURPHY, R. and S. R. WENTE, 1996 An RNA-export mediator with an essential nuclear export signal. Nature 383:357-360[Medline].
NAJARIAN, D., M. E. DIHANICH, N. C. MARTIN, and A. K. HOPPER, 1987 DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translation starts. Mol. Cell. Biol. 7:185-191
NISHIKURA, K. and E. M. DE ROBERTIS, 1981 RNA processing in microinjected Xenopus oocytes. J. Mol. Biol. 145:405-420[Medline].
PEEBLES, C. L., P. GEGENHEIMER, and J. ABELSON, 1983 Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 32:525-536[Medline].
PINES, J. and T. HUNTER, 1994 The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J. 13:3772-3781[Medline].
PRINGLE, J. R., A. E. M. ADAMS, D. G. DRUBIN and B. K. HAARER, 1991 Immunofluorescence methods for yeast, pp. 589–594 in Methods in Enzymology: Guide to Yeast Genetics and Molecular Biology, edited by C. GUTHRIE and G. R. FINK. Academic Press, San Diego.
REYNOLDS, A., V. LUNDBLAD, D. DORRIS and M. KEAVENEY, 1997 Yeast vectors and assays for expression of cloned genes, pp. 13.6.1–13.6.3 in Current Protocols in Molecular Biology, edited by F. M. AUSUBEL, R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN, J. A. SMITH and K. STRUHL. John Wiley & Sons, New York.
ROBBINS, J., S. M. DILWORTH, R. A. LASKEY, and C. DINGWALL, 1991 Two interdependent basic domains in nucleoplasmin nuclear targeting sequences: identification of a class of bipartite nuclear targeting sequences. Cell 64:615-623[Medline].
ROSE, A. M., H. BELFORD, W.-C. SHEN, C. GREER, and A. K. HOPPER et al., 1995 Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase. Biochimie 77:45-53[Medline].
SANGER, F., S. NICKLEN, and A. R. COULSON, 1977 DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467
SHOU, W., X. LI, C. WU, T. CAO, and J. KUANG et al., 1996 Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues. Mol. Cell. Biol. 16:990-997[Abstract].
SIKORSKI, R. S. and P. HIETER, 1989 A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.. Genetics 122:19-27
STOLC, V. and S. ALTMAN, 1997 Rpp1, an essential protein subunit of nuclear RNase P required for processing of precursor tRNA and 35S precursor rRNA in Saccharomyces cerevisiae.. Genes Dev. 11:2414-2425
TOLLERVEY, D. and T. KISS, 1997 Function and synthesis of small nucleolar RNAs. Curr. Opin. Cell Biol. 9:337-342[Medline].
VON HEIJNE, G., 1986 Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5:1335-1342[Medline].
WEN, W., J. L. MEINKOTH, R. Y. TSIEN, and S. S. TAYLOR, 1995 Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463-473[Medline].
WOLFE, C. L., Y.-C. LOU, A. K. HOPPER, and N. C. MARTIN, 1994 Interplay of heterogeneous transcription start sites and translational selection of AUGs dictate the production of mitochondrial and cytosolic/nuclear tRNA nucleotidyltransferase from the same gene in yeast. J. Biol. Chem. 269:13361-13366
WOLFE, C. L., A. K. HOPPER, and N. C. MARTIN, 1996 Mechanisms leading to and the consequences of altering the normal distribution of ATP(CTP):tRNA nucleotidyltransferase from the same gene in yeast. J. Biol. Chem. 271:4679-4686
ZOLADEK, T., G. VADUVA, L. A. HUNTER, M. BOGUTA, and B. D. GO et al., 1995 Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery. Mol. Cell. Biol. 15:6884-6894[Abstract].
This article has been cited by other articles:
 |
|
 |
|
  R. A. Haeusler and D. R. Engelke Spatial organization of transcription by RNA polymerase III Nucleic Acids Res., October 18, 2006; 34(17): 4826 - 4836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Butterfield-Gerson, L. Z. Scheifele, E. P. Ryan, A. K. Hopper, and L. J. Parent Importin-{beta} Family Members Mediate Alpharetrovirus Gag Nuclear Entry via Interactions with Matrix and Nucleocapsid J. Virol., February 15, 2006; 80(4): 1798 - 1806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Murthi and A. K. Hopper Genome-Wide Screen for Inner Nuclear Membrane Protein Targeting in Saccharomyces cerevisiae: Roles for N-Acetylation and an Integral Membrane Protein Genetics, August 1, 2005; 170(4): 1553 - 1560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. CHEKANOVA and D. A. BELOSTOTSKY Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export RNA, December 1, 2003; 9(12): 1476 - 1490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Hopper and E. M. Phizicky tRNA transfers to the limelight Genes & Dev., January 15, 2003; 17(2): 162 - 180. [Full Text] [PDF]
|
|
|
|
|
|
J. Lemieux, B. Lakowski, A. Webb, Y. Meng, A. Ubach, F. Bussiere, T. Barnes, and S. Hekimi Regulation of Physiological Rates in Caenorhabditis elegans by a tRNA-Modifying Enzyme in the Mitochondria Genetics, September 1, 2001; 159(1): 147 - 157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Maraia La Protein and the Trafficking of Nascent RNA Polymerase III Transcripts J. Cell Biol., May 14, 2001; 153(4): F13 - F18. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Azad, D. R. Stanford, S. Sarkar, and A. K. Hopper Role of Nuclear Pools of Aminoacyl-tRNA Synthetases in tRNA Nuclear Export Mol. Biol. Cell, May 1, 2001; 12(5): 1381 - 1392. [Abstract] [Full Text]
|
|
|
|
|
|
B. Gajewska, J. Kaminska, A. Jesionowska, N. C. Martin, A. K. Hopper, and T. Zoladek WW Domains of Rsp5p Define Different Functions: Determination of Roles in Fluid Phase and Uracil Permease Endocytosis in Saccharomyces cerevisiae Genetics, January 1, 2001; 157(1): 91 - 101. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. Kendall, M. W. Hull, E. Bertrand, P. D. Good, R. H. Singer, and D. R. Engelke A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing PNAS, November 2, 2000; (2000) 240454997. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Peng and J. E. Hopper Evidence for Gal3p's Cytoplasmic Location and Gal80p's Dual Cytoplasmic-Nuclear Location Implicates New Mechanisms for Controlling Gal4p Activity in Saccharomyces cerevisiae Mol. Cell. Biol., July 15, 2000; 20(14): 5140 - 5148. [Abstract] [Full Text]
|
|
|
|
|
|
T. Pederson and J. C. Politz The Nucleolus and the Four Ribonucleoproteins of Translation J. Cell Biol., March 20, 2000; 148(6): 1091 - 1096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Stanford, N. C. Martin, and A. K. Hopper SURVEY AND SUMMARY: ADEPTs: information necessary for subcellular distribution of eukaryotic sorting isozymes resides in domains missing from eubacterial and archaeal counterparts Nucleic Acids Res., January 15, 2000; 28(2): 383 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Vaduva, N. Martinez-Quiles, I. M. Anton, N. C. Martin, R. S. Geha, A. K. Hopper, and N. Ramesh The Human WASP-interacting Protein, WIP, Activates the Cell Polarity Pathway in Yeast J. Biol. Chem., June 11, 1999; 274(24): 17103 - 17108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kendall, M. W. Hull, E. Bertrand, P. D. Good, R. H. Singer, and D. R. Engelke A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing PNAS, November 21, 2000; 97(24): 13108 - 13113. [Abstract] [Full Text] [PDF]
|
|
- THIS ARTICLE
-
Abstract
- Full Text (PDF)
- Alert me when this article is cited
- Alert me if a correction is posted
- SERVICES
- Email this article to a friend
- Similar articles in this journal
- Similar articles in PubMed
- Alert me to new issues of the journal
- Download to citation manager
- Reprints & Permissions
- CITING ARTICLES
- Citing Articles via HighWire
- Citing Articles via Google Scholar
- GOOGLE SCHOLAR
- Articles by Tolerico, L. H.
- Articles by Hopper, A. K.
- Search for Related Content
- PUBMED
- PubMed Citation
- Articles by Tolerico, L. H.
- Articles by Hopper, A. K.