From the Institute of Applied Genetics, University of
Hannover, Herrenhäuser Strasse 2, D-30419 Hannover, Germany
and the § Laboratory of Eukaryotic Gene Regulation, NICHD,
National Institutes of Health, Bethesda, Maryland 20892-2716
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ABSTRACT |
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Based on characteristic amino acid sequences of
kinases that phosphorylate the subunit of eukaryotic translation
initiation factor 2 (eIF2
kinases), degenerate oligonucleotide
primers were constructed and used to polymerase chain reaction-amplify
from genomic DNA of Neurospora crassa a sequence encoding
part of a putative protein kinase. With this sequence an open reading
frame was identified encoding a predicted polypeptide with juxtaposed eIF2
kinase and histidyl-tRNA synthetase-related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast,
which stimulates translation of the transcriptional activator of amino
acid biosynthetic genes encoded by GCN4. Strains disrupted
for cpc-3 were unable to induce increased transcription and
derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to
up-regulate mRNA levels of cpc-1, encoding the
GCN4 homologue and transcriptional activator of amino acid
biosynthetic genes in N. crassa, but the mutation abolished
the dramatic increase of CPC1 protein level in response to amino acid
deprivation. These findings suggest that cpc-3 is the
functional homologue of GCN2, being required for increased
translation of cpc-1 mRNA in amino acid-starved cells.
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INTRODUCTION |
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In lower eukaryotes, like Neurospora crassa and
Saccharomyces cerevisiae, starvation for any one of a number
of amino acids leads to simultaneously induced transcription followed
by derepression of the enzymes in many amino acid biosynthetic
pathways. The global regulatory mechanism is referred to as general
amino acid control (discovered as "cross-pathway control" in
N. crassa, see Refs. 1 and 2). The ultimate element of the
signal transduction pathway, a transcriptional activator protein, is
encoded by the homologous genes, cpc-1 of N. crassa (3) or GCN4 of S. cerevisiae (4),
respectively. Recently, homologous proteins were reported also for
Aspergillus niger and Cryptonecria parasitica (5, 6). In yeast, GCN2 plays a crucial role in signal perception and transduction. GCN2 encodes a protein containing an
eIF21 kinase domain (7-9)
that is required for increased GCN4 protein synthesis in amino
acid-starved cells.
eIF2 kinases regulate initiation of protein synthesis (10) by
phosphorylation of the
subunit of eukaryotic translation initiation
factor 2 (eIF2
) on Ser-51. GTP-bound eIF2 is necessary for
delivering charged initiator tRNAMet
(Met-tRNAiMet) to the 40 S ribosomal
subunits, and after initiation of translation it is released as
eIF2·GDP. The phosphorylated form of eIF2 sequesters its own
recycling factor eIF2B necessary for exchange of GDP by GTP (11). As
only the GTP-bound form of eIF2 is able to initiate translation,
sequestering of eIF2B leads to a general reduction of protein
synthesis. However, activation of GCN2 in yeast leads to increased
translation of one mRNA species, GCN4 mRNA. This gene-specific regulation is mediated by four short upstream open reading frames (uORF) in the 5' leader of GCN4 mRNA
(4).
Extensive genetic analysis of the GCN4 mRNA leader has
provided a detailed model for GCN4 translational regulation
(4). Irrespective of amino acid availability, the first uORF is
translated, and about 50% of the ribosomes resume scanning on the
mRNA. Under non-starvation conditions translation of the following
three uORFs leads to dissociation of almost all the ribosomes from the
mRNA due to specific sequences surrounding the translational stop
codons, and therefore translation of GCN4 is prevented.
Under amino acid starvation conditions GCN2 becomes activated and
phosphorylates eIF2, leading to low levels of GTP-bound eIF2 and,
therefore, reduced concentration of
eIF2·GTP·Met-tRNAiMet ternary
complexes. Consequently, after translation of uORF1, ribosomes resume
scanning, but the rate at which they rebind ternary complexes is
lowered. Thus, ribosomes are less able to re-initiate at any of the
translation initiation sites of the following three uORFs, and many
re-initiate at GCN4 instead.
So far three eIF2 kinases are known that share extensive homology in
the kinase catalytic domain. Apart from the 12 conserved subdomains
found in most protein kinases, they have additional characteristic
features, including an insert between subdomains IV and VI and
subdomains IX and X, respectively, which distinguishes them from other
serine/threonine kinases (10, 12). However, each of these kinases are
activated by distinct stimuli as follows: the heme-regulated inhibitor
(HRI) in rabbit and rat by heme deficiency (13, 14), the
double-stranded RNA-dependent kinase (PKR) in human, mouse,
and rat by the occurrence of double-stranded RNAs after virus infection
(15-17), and GCN2 of S. cerevisiae by amino acid
deprivation. The activation signal and target for the recently discovered Drosophila melanogaster homologue of yeast GCN2,
DGCN2, are not known (18). In addition to the kinase catalytic domain, each eIF2
kinase contains unique sequences that may be responsible for its own characteristic regulation. For example PKR contains two
double-stranded RNA-binding motifs required for RNA binding (19, 20).
Within the kinase catalytic domain of HRI, two heme regulatory motifs
are known (21, 22). Adjacent to the eIF2
kinase catalytic domain,
GCN2 contains a domain that resembles the histidyl-tRNA synthetases
(HisRS), which was postulated to monitor amino acid availability
(8).
Early work by various N. crassa and yeast researchers (23)
indicated that uncharged aminoacyl-tRNAs that accumulate in amino acid-deprived cells are the relevant signal in the mechanism of general
control. Mutations in the HisRS-like domain of GCN2 were found to
impair phosphorylation of Ser-51 of eIF2 and the derepression of
GCN4 mRNA translation in amino acid-starved cells. Wek
et al. (9) could demonstrate binding of uncharged tRNAs to
the synthetase-related domain. The exact interaction between the GCN2
regulatory and catalytic domains upon activation is not yet known. The
N-proximal domain containing a degenerate protein kinase
moiety (8, 24) and the C-terminal region beyond the HisRS-like domain
are also required for GCN2 function (25). For the latter,
Ramirez et al. (25) demonstrated a function in ribosome
association of the protein and a role in dimerization was recently
elucidated as well (89).
In contrast to yeast, where GCN2 and several other elements were identified genetically by abundant mutations that impair general amino acid control, all but one of the regulation-deficient mutations of N. crassa mapped in the cpc-1 gene (26-28). The one exception was a mutation that identified the cpc-2 gene (30, 31); however, cpc-2 of N. crassa showed no relationship with any of the known yeast genes involved in general amino acid control. We were interested, therefore, to find out whether substantial differences exist in the details of the mechanism of amino acid regulation between these ascomycetes and searched for a N. crassa gene with homology to yeast GCN2.
Here the molecular identification of the N. crassa cpc-3 gene and its characterization as a structural homologue of yeast GCN2 is reported. The molecular engineering of a cpc-3 disruption allele and the phenotypic consequences of the loss of function are described. Our results show that the cpc-3 product is a positive regulator of the general control response of N. crassa and most likely functions as a translational activator of cpc-1, analogous to the function of yeast GCN2.
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EXPERIMENTAL PROCEDURES |
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Strains and Culture Conditions--
The N. crassa
wild-type strain (St. Lawrence 74A) and the strains cyh-2,A,
arg-12s,a arg-12,a
were obtained from the Fungal Genetics Stock Center (FGSC, University
of Kansas Medical Center); the cpc-1(j-5) and
cpc-2(U142) mutant strains were from the Barthelmess
lab.
Plasmids and Libraries-- The ordered N. crassa genomic cosmid library of Vollmer and Yanofsky (33) was used to screen for cpc-3 sequences. Plasmids used in this study were pCPC1-2 for cpc-1 (3), pCPC2-C8 for cpc-2 (31), arg-12 in pUC8 (34), pACTIN for the actin encoding gene (M. Plamann), pBT6 for the Bml cassette (35), and pCSN43 for the hph cassette (36).
DNA fragments were cloned, and the cpc-3 disruption allele was constructed in pBluescript SK. PCR amplification products were cloned in pUC19. E. coli strains used were DH1 for the cosmid library and XL-1 blue for all other purposes. Transformation of E. coli was carried out according to Mandel and Higa (88) or, in case of plasmids larger than 10 kb, via electroporation (37, 38).Transformation of N. crassa-- Spheroblasts obtained from germinating conidia were used for transformation (33). Transformants were made homokaryotic via the isolation of microconidia-derived colonies (39).
Isolation and Analysis of DNA-- Isolation of high quality and pure genomic DNA from N. crassa followed the method of Lee et al. (40). For PCR analysis of large numbers of genomic DNA samples the methods of Irelan et al. (41) and Chow and Kaefer (42) were combined as follows: N. crassa was incubated for 2 days in 1 ml of stagnant liquid culture. Mycelia were squeezed between Whatman paper and transferred to 0.2 ml of isolation buffer (0.2 M Tris-HCl, 0.5 M NaCl, 0.01 M EDTA, 1% SDS, pH 7.5). After addition of glass beads (0.3-0.4 mm diameter) and 0.2 ml of 1:1 phenol:chloroform the samples were vortexed for 5 min followed by addition of 0.3 ml of isolation buffer and 0.3 ml of phenol:chloroform and centrifugation (30 s, 5000 × g). The liquid phase was again extracted with 0.3 ml of phenol:chloroform. The DNA was precipitated with 1 ml of ethanol, dissolved in 100 µl of TE buffer (containing 100 µg/ml RNase) at 37 °C for about 1 h, ethanol-precipitated, and finally dissolved in 50 µl of TE buffer.
Southern analysis followed standard protocols (43) using nylon membranes (Amersham Pharmacia Biotech). Probes were labeled with DIG-11-dUTP (DIG-DNA random primed labeling kit, Boehringer Mannheim). Labeling of DNA shorter than 700 bp was performed by PCR reaction (see below, except thatScreening of the Genomic Library-- Clones of each microtiter plate of the N. crassa ordered genomic cosmid library (33) were pooled, and pure DNA was isolated (plasmid midikit, Qiagen). By using 1 µg DNA of each pool, a dot blot membrane was generated and screened using cpc-3-specific sequences as probes (hybridization technique as described for Southern analysis). To identify the individual positive clones, colonies of each microtiter plate of interest were transferred to solid medium with a microtiter replica plater and subjected to colony hybridization (43).
RNA Isolation and Northern Blot Analysis--
Isolation of total
cellular RNA and preparation of Northern blots were done according to
Sokolowsky et al. (44) using 10 µg of RNA of each sample
and nylon membranes (N+, Amersham Pharmacia Biotech). Probing was done
according to Sambrook et al. (43). DNA probes were
radiolabeled with [-32P]dCTP (random-primed labeling
kit, Life Technologies, Inc.) and purified on Sephadex columns (43).
Probes were stripped from membranes by washing with 5% (w/v) SDS at
65 °C for at least 10 min.
Nucleotide Sequence Analysis-- By using PCR, DNA sequences were determined by the Sanger dideoxy sequencing method (fmol sequencing kit, Promega). A 1.6-kb SmaI-EcoRI fragment of cpc-3 was commercially sequenced (LARK). DNA sequences were analyzed with programs DNASIS and PROSIS (Hitachi). Predicted amino acid sequences were compared with available protein sequences using the basic local alignment search tool (BLAST, see Ref. 45). Multi-alignments were performed using the GCG program (46).
Enzyme Assays-- All assays were performed with crude extracts from freeze-dried mycelia. The specific activities of L-ornithine carbamoyltransferase (EC 2.1.3.3) and citrate-synthase (EC 4.1.3.7) were assayed according to Davis (47) or Flavell and Fincham (48), respectively.
Protein Isolation and Immunoblotting-- Crude cell extracts of N. crassa were isolated by grinding fresh mycelium in liquid nitrogen, addition of equal volumes of breaking buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.2% Triton, 1 tablet of protein inhibitor mixture (complete, Boehringer Mannheim) per 50 ml of buffer, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM dithiothreitol) and of glass beads (0.3-0.4 mm diameter), and subsequent vortexing 6 times for 30 s at 4 °C. After removal of cell debris (14,000 rpm, 10 min, 4 °C), Western blots were conducted by using precast gels and nitrocellulose membranes (NOVEX) according to the manufacturer's protocol. Detection of antigen-antibody complexes was performed by using horseradish peroxidase-conjugated anti-rabbit antibodies and the enhanced chemiluminescent detection system (Amersham Pharmacia Biotech).
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RESULTS |
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Molecular Identification of the N. crassa cpc-3 Gene--
Based on
the amino acid sequences in the catalytic domains of eIF2 kinases
(8, 13, 15, 16), highly conserved groups of amino acids in the insert
region between kinase subdomains IV and VI (characteristic of eIF2
kinases) and in subdomain VII were chosen for the construction of
degenerate oligonucleotides (called 2.1 and 2.3, Table I). The oligonucleotides bracketed subdomain VI that contains amino acids characteristic of
serine/threonine protein kinases and is surrounded by amino acids
typical of eIF2
protein kinases. Knowledge of the sequence of PCR
fragments amplified with these primers should be sufficient to
determine whether or not they derived from a gene encoding an eIF2
kinase. By using genomic N. crassa DNA as template, a single
302-bp PCR product, called 2.1-2.3, was obtained using 2.1 and 2.3 as
primers (Fig. 1B) that encodes
an amino acid sequence with 60% sequence identity to the corresponding
yeast GCN2 segment.
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Genomic Organization of the cpc-3 Locus-- cpc-3 is 5162 bp in length and consists of 5 exons totaling 4941 bp of cpc-3 coding region. The 4 introns were identified by the conserved splice junctions and lariat sequences (54, 55) and confirmed by RT-PCR reactions using intron flanking primers (not shown). Intron positions did not coincide with the domain structure of the cpc-3-encoded polypeptide. GCN2 lacks introns (8). For DGCN2 only cDNA sequences have been reported (18).
The putative translation start point was narrowed down via the determination of the most 5' in-frame stop codon. Sequences surrounding the first downstream ATG codon showed the best match to the N. crassa Kozak consensus sequence as compared with further downstream ATG codons (54, 55). RT-PCR analysis verified that this putative translational start codon and the sequences upstream of it were part of the cpc-3 mRNA (Fig. 2). This also indicated that the 5' leader sequence is at least 220 bp in length (Fig. 2), which is unusually long for filamentous fungi (53). The sequence (TGTATTA) 77 bp downstream from the TGA codon may represent a polyadenylation signal (AGTATAA, see Refs. 53 and 54). The length of the transcription unit is at least 5238 bp, in agreement with the length of the observed faint transcript (6 kb).
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Comparative Analysis between N. crassa CPC3 and the Yeast and
Drosophila GCN2 Polypeptides--
The deduced amino acid sequence of
1646 amino acids showed the highest overall similarity to GCN2 of yeast
(Ref. 8, translation start site according to Ref. 8 and accession
number U51030) and DGCN2 of Drosophila (18) (31% identity
between GCN2 and DGCN2), with 35 and 32% positional identity,
respectively, over almost the entire length of the
cpc-3-encoded polypeptide (Fig. 3). Only about 30 amino acids at the N
terminus of CPC3 were exempt from the alignment, i.e. CPC3
was found to be longer than Drosophila or yeast GCN2 (8,
18), respectively. The high similarity between the proteins is
highlighted when the comparison includes equivalent amino acids
(PROSIS); at 58/54% of the positions of GCN2/DGCN2 either identical or
equivalent amino acids were found in CPC3 (54% between GCN2 and
DGCN2). The similarity between the proteins allowed us to distinguish
for CPC3, as for the GCN2 proteins, four regions/domains with
characteristic features: the eIF2 kinase and histidyl-tRNA
synthetase-like (HisRS-like) domains and the N- and C-terminal regions
(Fig. 1D):
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Construction of a cpc-3 Mutation, cpc-3::hph, via
Homologous Genomic Integration of an in Vitro Constructed Gene
Disruption--
To study the function of cpc-3 a putative
loss of function mutation was engineered via a plasmid-borne
cpc-3 disruption construct (Fig.
6). The strategy involved the deletion of
about 1 kb of the cpc-3 gene, including the region encoding
subdomains VI-XI of the eIF2 kinase domain and part of motif 2 of
the HisRS-like domain, and replacing them by the hph
cassette as a dominant selectable marker conferring resistance to
hygromycin B. A homologous double recombination event was required for
replacement of the wild-type cpc-3 allele by the
plasmid-borne disruption construct (Fig.
7) which is a rare event in filamentous
fungi. To enable a rapid screen of transformants that were likely to
contain gene replacements, the Bml cassette was inserted 3'
of the cpc-3::hph allele on the plasmid (Fig. 6).
N. crassa Bml encodes a benomyl-resistant
-tubulin, providing a dominant marker which should be lost in the course of
homologous recombination (67) (Fig. 7). However, since ectopic integration of parts of the plasmid could equally result in
benomyl-sensitive transformants, molecular proof for correct gene
replacement was required. By using one primer (11.2) complementary to
hph sequences and another (11.1) complementary to sequences
5' of the cpc-3 disrupted region (present in the host genome
but missing in the transforming plasmid), a PCR amplification product
of 2.1 kb should be produced if genomic cpc-3 is replaced by
cpc-3::hph via homologous recombination (Fig. 7).
Homokaryotic cpc-3::hph strains should not yield
amplification of the 302-bp PCR fragment with primers 2.1 and 2.3, where 2.3 is complementary to the deleted region of cpc-3
(Fig. 7). Further confirmation for site-specific and unique integration
was obtained by Southern analysis and genetic linkage studies (see
below).
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cpc-3 Disruption Interferes with the Regulation of Amino Acid Biosyntheses-- Since the homokaryotic cpc-3::hph mutant strains were not only viable but both grew and reproduced vegetatively and sexually like the wild type, we concluded that cpc-3 does not provide an essential cellular function. The structural homology between CPC3 and GCN2 called for a closer examination of amino acid regulation in cpc-3::hph mutants (abbreviated cpc-3). Starvation for histidine was achieved by supplementing the medium with 3AT, a competitive inhibitor of imidazole glycerophosphate dehydrogenase in histidine biosynthesis (68). N. crassa wild type with intact amino acid regulation can grow on certain 3AT concentrations; however, regulation deficient mutants like N. crassa cpc-1 and cpc-2, unable to counteract enzyme inhibition via derepression of amino acid biosynthetic enzymes, are 3AT-sensitive (27, 30).
Homokaryotic cpc-3 mutants derived from either S1/152 or S1/148 were found to be 3AT-sensitive (simultaneous supplementation with histidine-restored growth). 3AT sensitivity was recessive in cpc-3/cpc-3+ heterokaryons. In crosses between cpc-3 mutants and wild type, the 3AT sensitivity and hygromycin resistance phenotypes were tightly linked and did not separate (not shown), indicating a causal relationship between the cpc-3 disruption and the defect in the regulation of histidine biosynthesis. Any combination of forced heterokaryons carrying two different nuclei with mutations in cpc-3, cpc-1, or cpc-2, respectively, showed complementation of the 3AT sensitivity (not shown), confirming that these mutations identify different functions. To obtain evidence that the cpc-3 mutant had a "cross-pathway" defect, we investigated the regulation of the arginine biosynthetic enzyme L-ornithine carbamoyltransferase (coded for by arg-12 in N. crassa) in response to histidine deprivation imposed by 3AT supplementation. Fig. 9 shows that a 5-fold induction of enzyme activity (derepression) occurred in the wild-type, the cyh-2 recipient, and the cpc-3::hph/cpc-3+ heterokaryotic strains. However, a complete lack of enzyme derepression was found in all homokaryotic cpc-3::hph subcultures in response to growth on 3AT. The remaining enzyme level in the mutants was similar to the uninduced wild-type activity, comparable to the phenotype of cpc-2 mutants (30), whereas cpc-1 mutants cause a further reduction in basal enzyme level (27) (Fig. 9). Functional consequences of the observed basal enzyme activity were investigated by introducing the regulatory mutations into the arg-12s background. The bradytrophic arg-12s allele encodes for an enzyme with drastically reduced OCT activity (47). An arg-12s;cpc-3 double mutant was found to grow almost at the wild-type rate without arginine supplementation (like arg-12s;cpc-2, see Ref. 30), whereas an arg-12s;cpc-1 strain is an arginine auxotroph (26, 27) (data not shown). This suggested that in a cpc-3 mutant the basal level of cpc-1 function provides sufficient induction of arg-12s transcript for arginine prototrophy and that the cpc-3::hph mutation does not decrease the basal enzyme activity of enzymes under general amino acid control.
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cpc-3 Is a Posttranscriptional Activator of cpc-1 Expression-- In cpc-3 mutants we found that amino acid starvation leads to an increase in cpc-1 mRNA level but not to derepression of arg-12 transcription, a target gene of CPC1. To obtain additional evidence that cpc-3 functions at a posttranscriptional step to increase cpc-1 expression in amino acid-starved cells, we studied CPC1 protein levels (Fig. 11). Consistent with previous observations (49) CPC1 is undetectable under non-starvation conditions but readily detectable under amino acid deprivation. In contrast, the cpc-3 mutant did not show any detectable CPC1 under starvation conditions. Because CPC1 is undetectable in starved cpc-3::hph mycelium, it is impossible to calculate the reduction in CPC1 expression conferred by the cpc-3 mutation. However, we estimate that the CPC1 level is at least 10-fold greater in the wild-type versus cpc-3 mutant, a much greater difference than the 1.7-fold higher amount of cpc-1 mRNA in wild type. These findings support the idea that cpc-3 is a translational activator of cpc-1 expression.
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DISCUSSION |
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The conservation of the polypeptide sequence found between
N. crassa CPC3, yeast GCN2, and Drosophila GCN2
argues that these are homologous proteins. The most notable structural
similarity is the juxtaposition of a protein kinase and HisRS-related
domain. In addition, GCN2 and CPC3 share extensive similarity in
degenerate kinase domains located N-terminal to their conventional
kinase domains. The CPC3 kinase domain contains many of the conserved features observed previously for the eIF2 kinases GCN2, HRI, and PKR
(57). The only continuous amino acid stretches uniquely conserved in
the GCN2-like proteins are
"WRLFRXIXEXL" in subdomain VIA
and "VVRY" in subdomain IV. The CPC3 HisRS-like domain lacks sequences essential for binding both histidine and ATP, supporting the
model that the HisRS-like domains in the GCN2-related kinases lack
HisRS activity and function as sensors of multiple uncharged tRNAs (8).
It was shown that the HisRS-related domain of yeast GCN2 binds
uncharged tRNA (9) and that the HisRS-like sequences are required for
in vitro activation of GCN2 kinase function (66); however,
it remains to be shown that discrimination between tRNA species is
lacking. Consistent with nonspecific binding of tRNAs, as noted for
GCN2 (57), motif 2 sequences for the class II enzyme AspRS that
interact with the 3' end of the acceptor stem of yeast tRNAAsp (63) are conserved in genuine HisRS but only
partially conserved in CPC3 and DGCN2. In addition, the C-terminal
domain of E. coli HisRS was shown to be responsible for
recognition of the tRNAHis anticodon (70), and the only
HisRS-characteristic motif in this region (Ref. 62 and Fig. 5), is
poorly conserved in the HisRS-like domains of the GCN2-related
kinases.
Is there an explanation for the choice of HisRS to be linked in
evolution to the eIF2 kinase domains of the GCN2-like proteins? The
unique ability of HisRS to recognize acceptor stem base pairs both in
the context of full-length tRNA and in mini- or microhelices (73-76)
might single out this enzyme as the best candidate for diversification
of tRNA binding specificity. Monitoring uncharged tRNAs in general
would require that the HisRS-like domains ignore the unique identity
element of tRNAHis species, the extra nucleotide
G
1, at their 5' end (77).
Since the disruption mutation of cpc-3 destroyed essential parts of the kinase and HisRS-like domains, a complete loss of function was assumed. The phenotypes of the cpc-3::hph mutant proved that the gene is required for the function of general amino acid control. cpc-3 mutations were probably not detected in searches for N. crassa regulatory mutations since most of these relied on the postulated arginine auxotrophy of cpc mutations in an arg-12s background (26, 27). We found that arg-12s;cpc-3 double mutants grew in unsupplemented medium.
The extensive structural similarity to GCN2 suggested that CPC3 has a
function in general amino acid control equivalent to that of GCN2,
namely translational activation of cpc-1, the
GCN4 homologue of N. crassa. Consistent with this
conclusion, cpc-1 mRNA becomes associated with larger
polysomes after transfer of N. crassa to histidine
starvation medium (71), and there are two uORFs in the cpc-1
mRNA leader (see Ref. 3, accession number J03262). The 5' leader of
GCN4 mRNA contains four uORFs necessary for
gene-specific translational activation of GCN4 expression by
GCN2, but the first and fourth uORFs are sufficient for almost wild-type regulation (4). GCN4 translational control
requires that the first uORF does not promote dissociation of ribosomes after termination of translation, and the last codon and 10 bases 3' to
the translational stop codon are decisive for this property (72). As
mentioned by Luo et al. (71), the nucleotide composition and
C/G content around the stop codons of cpc-1 uORF1 and uORF2 are similar to those at GCN4 uORF1 and uORF4, respectively,
suggesting a common translational mechanism for GCN4 and
cpc-1. In agreement with the idea that CPC3 is a
translational activator of cpc-1, we found that amino acid
deprivation in a cpc-3 mutant did not lead to any detectable
increase in CPC1 protein level, despite a remarkable increase in
cpc-1 mRNA levels. From this we propose that CPC3
stimulates translation of cpc-1 mRNA by the same
mechanism elucidated for GCN4 mRNA in yeast, involving
down-regulation of eIF2·GTP·Met-tRNAiMet ternary
complex formation by phosphorylation of eIF2.
If CPC3 stimulates cpc-1 mRNA translation by the same
mechanism elucidated for GCN2/GCN4 mRNA in yeast (4), we
would expect to observe increased phosphorylation of eIF2 in amino
acid-starved N. crassa cells. By using isoelectric focusing
gels, increased phosphorylation of eIF2
under amino acid deprivation
was shown in yeast (83). Because antibodies against N. crassa eIF2
are not available, and the yeast eIF2
antibodies
do not appear to cross-react with the N. crassa protein
(data not shown), we could not test this prediction. The sequences
surrounding Ser-51 in yeast eIF2
, the phosphorylation site
recognized by GCN2, PKR, and HRI (84), are highly conserved between
yeast, mammals, and Drosophila (85); however, the sequence
of N. crassa eIF2
is not known. For the eIF2
kinase
domains of PKR and GCN2, it was found that phosphorylation of two Thr
residues in the activation loop are required for high level kinase
activity (86). In CPC3 these Thr residues are conserved (Fig. 4)
suggesting a similar activation/regulation mechanism as for GCN2 and
PKR.
Thus far the investigation of N. crassa cpc-3 does not point out distinct differences in the mechanism of general control between N. crassa or yeast. Comparable to the yeast system (78, 79), induction of cpc-1 mRNA in response to amino acid limitation occurred not only in the presence of the cpc-3 mutation (this investigation) but also in the presence of various cpc-1 alleles (3, 29). This argues that an independent second mechanism must exist that can register amino acid deprivation and stimulate cpc-1 transcription.
A search in the EST data base identified sequence fragments of mouse
and human covering a stretch from protein kinase subdomain XI to the
N-terminal part of HisRS sequences (up to motif 2 for mouse EST
accession number AA016507; human EST accession number AA216651)
suggesting that mammals possess an eIF2 kinase linked to a
HisRS-like domain. This might indicate a general metabolic requirement
for eIF2
kinases activable by uncharged tRNA, providing the means to
down-regulate general translation and induce a starvation response
protein like CPC1 or GCN4. N. crassa cpc-3 or yeast
gcn2
mutants do not show any restriction in vegetative
growth or sexual reproduction under non-starvation conditions,
indicating that CPC3 and GCN2 are not critically involved in these
processes. The developmentally regulated expression of DGCN2 and, in
later stages, restricted expression in a few cells of the central
nervous system (18) suggest the exciting possibility of additional
functions for this interesting protein kinase in higher organisms.
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ACKNOWLEDGEMENTS |
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We thank Robert Metzenberg and Ronald Wek for helpful comments; Michael Plamann for the N. crassa actin gene; and Charles Yanofsky for the anti-CPC1 antiserum.
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FOOTNOTES |
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* The work was supported in part by a grant from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X91867.
¶ Recipient of a National Research Council Research Associateship. To whom correspondence should be addressed: Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bldg. 6A, Rm. B1A-05, Bethesda, MD 20892-2716. Tel.: 301-435-6023; Fax: 301-496-8576; E-mail: esattlegger{at}aghmac1.nichd.nih.gov.
The abbreviations used are: 3AT, 3-amino-1,2,4-triazole; eIF, eukaryotic translational initiation factor; HRI, heme-regulated inhibitor; PKR, double-stranded RNA-dependent kinase; GCN, general amino acid control non-derepressible; CPC, cross-pathway control; PCR polymerase chain reaction, uORF, upstream open reading frame; bp, base pair(s); kb, kilobase pair(s); HisRS, histidyl-tRNA synthetase(s); RT, reverse transcriptase; DIG, digoxigenin.
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