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INTRODUCTION |
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
eIF2
1 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 |
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.
N. crassa cultivation on Vogel's standard medium and
crossing techniques followed Davis and de Serres (32). Briefly, for enzyme assays and DNA, RNA, and protein isolation, exponentially grown
mycelium was obtained by inoculation of 100 ml of liquid medium with
0.5-1 × 106 conidia and incubation overnight at
29 °C and 170 rpm. For growth tests 1 ml of stagnant liquid medium
was inoculated with mycelial slants and incubated at 29 °C.
If required, Vogel's medium was supplemented with final concentrations
of 0.5 µg/ml benomyl, 1 µg/ml cycloheximide, 250 or 333 µg/ml
(liquid or solid medium) hygromycin B, 4 or 6 mM (stagnant liquid or exponential culture) 3-amino-1,2,4-triazole (3AT), and 40 mM acetate (omitting glucose), respectively. All
supplements were prepared as stock solutions, sterile-filtered, and
added to the autoclaved medium.
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 that 
of dTTP was replaced by DIG-dUTP), and
the labeling reaction was used directly for hybridization.
Hybridization and detection of probes and stripping of probes from
membranes followed the manufacturer's protocol (DIG luminescent
detection kit, Boehringer Mannheim).
PCR reaction mixtures consisted of 1× PCR buffer (10 mM
Tris-HCl, 1.5 mM MgCl2, 50 mM KCl,
pH 8.3, Boehringer Mannheim), 200 µM of each dNTP, 500 nM each primer, 0.02 units/µl Taq polymerase (Perkin-Elmer or Boehringer Mannheim), and 5 ng/µl genomic DNA or 5 pg/µl plasmid/cosmid DNA. PCR of 10-100-µl volumes were performed in a Perkin-Elmer DNA thermal cycler TC1; the cycles were 30 s at
95 °C (5 min in the first cycle), 1 min at the annealing temperature (5 °C lower than Tm, for degenerate primers see
Table I). Extension time was 1 min per 1 kb at 72 °C.
For RT-PCR analysis cDNA was synthesized from 1 µg of total RNA
using Superscript RT RNase H reverse transcriptase (Life
Technologies, Inc.) according to the manufacturer's protocol. Aliquots
of the reverse transcription reaction mixture (10% (v/v) of final PCR
reaction) were directly subjected to PCR reactions.
Screening 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 |
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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Table I
Oligonucleotide sequences used for PCR reactions
Lowercase letters indicate nucleotides added at the 5' end for the
construction of restriction sites (underlined). (Numbering of the
nucleotide positions refers to the translation start point of
cpc-3.) The PCR primers were tested for suitability using
the program OLIGO (TIB molbiol). The annealing temperatures used for
degenerated primers are indicated. s, sense primer, a, antisense
primer. Letters indicating variability are Y (C or T), I (inosine), R
(A or G), and H (A or C or T).
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Fig. 1.
Molecular organization of the N. crassa
cpc-3 gene, subcloned fragments, and domain structure of the
encoded polypeptide. The positions of eIF2 kinase subdomains I,
VI, and VII and the positions of motifs 1-3 in the HisRS-like domain
are indicated. A, restriction map indicating the exon/intron
structure; cpc-3 is indicated by a black bar and
introns are shown in white. B, PCR amplification
products obtained from genomic DNA (2.1-2.3, 4.1-4.2) or cosmid clone
17:5D (5.1-5.2, 6.1-6.2). C, subcloned restriction
fragments from cosmid clone 17:5D. D, domain structure of
CPC3 (accession number X91867), GCN2 (U51030), and DGCN2 (U80223) in
relation to amino acid positions (8, 18).
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The 2.1-2.3 PCR product was used as a probe to isolate two cosmid
clones (17:5D and 20:1F) from an ordered genomic library (33). By using
cosmid 17:5D as template, and in each case a specific primer
constructed from already sequenced areas, and a degenerate primer (see
below), adjacent overlapping stretches of DNA were synthesized by PCR.
Degenerate primer 4.1 was constructed according to characteristic amino
acids of protein kinase subdomain I. Construction of degenerate primers
5.2 and 6.2, respectively, was guided by conserved sequences of HisRS
proteins. Sequencing of the PCR-amplified fragments 4.1-4.2, 5.1-5.2,
and 6.1-6.2 (Fig. 1B) indicated that N. crassa
contains a GCN2-like gene (i.e. encodes a protein
characterized by juxtaposed kinase and HisRS-like domains).
From PCR fragments and subcloned restriction fragments of cosmid 17:5D
(Fig. 1C), a restriction map was derived (Fig.
1A). Sequencing of PCR fragments and subcloned restriction
fragments or direct sequencing of cosmid 17:5D DNA led to the
determination of a DNA sequence with coding capacity for a GCN2-like
polypeptide (Ref. 50, accession number X91867).
By using 2.1-2.3 (Fig. 1B) for restriction fragment length
polymorphism studies (51, 52), the corresponding N. crassa gene, cpc-3, was located on the right arm of linkage group V
close to cyh-2 (4.2% linkage). Southern hybridization with
the 2.1-2.3 DNA probe or with larger DNA segments (4.1-4.2 and
6.1-6.2, respectively) suggested that cpc-3 represents a
single copy sequence (not shown).
By using the 2.1-2.3 fragment, or larger cpc-3 sequences,
as a probe in Northern experiments, we observed a faint signal of about
6 kb in total RNA, indicating a low abundance mRNA. However, using
RT-PCR methods the expression of cpc-3 was unambiguously demonstrated (not shown). Low expression of cpc-3 is
suggested by the codon usage which is typical for low and
non-constitutively expressed genes (53).
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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Fig. 2.
Confirmation of the putative N. crassa
cpc-3 translational start site as part of the cpc-3
transcript using RT-PCR analysis. Reverse transcription on
N. crassa total RNA with primer 14.2 (Table I), located 3'
from intron 1, was followed by PCR amplification using the primer pair
14.1/14.2 (flanking intron 1, with 14.1 representing the most 5'
available sequence, i.e. located 220 bp upstream of the
putative translational start site) yielding a fragment of 822 bp
(lane 1). As control, the same primer pair was used in a PCR
reaction on genomic DNA as template, resulting in a 879-bp fragment
(lane 2).
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Sequence analysis of the upstream sequences of cpc-3,
GCN2, and DGCN2 showed no remarkable homologies,
and whereas all three contain uORFs, their positions are not conserved.
Located 136 bp upstream of cpc-3 is an uORF coding for 5 amino acids; however, as the whole sequence of the 5' leader is not
known its translational start site might be further upstream. Two uORFs
also are present upstream of DGCN2 (distance from
DGCN2, 38 and 95 bp). uORFs were found that overlap with the
translational start points of DGCN2 and GCN2 but
not cpc-3. If the uORFs are involved in translational regulation, the expression of the GCN2-like proteins might be differently regulated.
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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Fig. 3.
Multiple sequence alignment of N. crassa CPC3 (accession number X91867), yeast GCN2 (Ref. 6,
U51030), and Drosophila DGCN2 (Ref. 15, U80223) using GCG
program PileUp (gap creation penalty 6, gap extension penalty 2), with
some manual adjustment in areas of low similarity. The N- ( )
and C-terminal ( ) ends of the CPC3 N-terminal domain, eIF2
kinase (PK domain), HisRS-like, and C-terminal domains are indicated.
In the N-terminal domain, amino acids written in bold
letters represent positions matching with kinase-characteristic
sequences. Kinase subdomains (roman numerals) and
C-/N-terminal boundaries, their characteristic amino acids including
tyrosine (1) and serine/threonine kinase-specific sequences
(2) are indicated above the alignment of the
degenerate kinase region (12, 56, 80). Positions conserving nonpolar
residues (§, FYWIMVLA), polar residues (±, HRKDENQ), small residues
with near neutral polarity (", PGST), and aromatic residues
(o, YFW) are indicated. Lowercase letters
represent modest conservation of the corresponding amino acids,
capital letters high conservation, and bold capital
letters invariant residues. The consensus sequence marks positions
with 100% (capital letters) or 67% identity (small
letters) among the GCN2-like proteins.
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The highest sequence conservation was observed in the kinase domain
(amino acids 597-971, Fig. 4), among
CPC3, GCN2, and DGCN2 with 46/42/41% (CPC3 versus GCN2/CPC3
versus DGCN2/GCN2 versus DGCN2) identity and
64/61/60% similarity. This domain in CPC3 contained all the invariant
and most of the highly conserved amino acids found in the subdomains of
protein kinases (12, 56) (Fig. 4). Based on the sequences of subdomains
VI and VIII, it is expected to be a serine/threonine kinase. CPC3 bears
a large insert of about 120 amino acids between subdomains IV and VI
typical of eIF2 kinases. It also contains most of the signature
amino acids of eIF2 kinases diagnosed by Ramirez et al.
(57), plus eIF2 kinase-specific conservation in the areas of
subdomains IX and X (14) (Fig. 4). These findings suggested that
cpc-3 encodes a functional eIF2 serine/threonine
kinase.
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Fig. 4.
Multiple sequence alignment of all known
eIF2 kinase domains (Program GCG, gap creation penalty 10, gap
extension penalty 2: PKR from rat (rPKR, accession number
L29281), mouse (mPKR, accession number Q03963), and human
(hPKR, accession number P19525); HRI from rat
(rHRI, accession number L27707) and rabbit
(kHRI, accession number P33279); GCN2-like proteins from
N. crassa (CPC3, accession number X91867),
S. cerevisiae (GCN2, accession number U51030),
and Drosophila (DGCN2, accession number
U80223). Below, the consensus sequence of PKR, HRI, and
GCN2-like (GCN2l) proteins and all eIF2 kinases (called
consens) are shown, respectively. Conservation of an amino
acid of at least 50% in each kinase group is shown in lowercase
letters, and absolute identities are indicated with capital
letters. The 11 eIF2 kinase-characteristic amino acids pointed
out for GCN2 by Ramirez et al. (57) are indicated by  . In
GCN2 between subdomains VII and VIII, the autophosphorylation sites
required for kinase activity (Thr-882, Thr-887, Ref. 86) are
underlined. Kinase subdomains (roman numerals)
and their characteristic amino acids (12) are indicated
above the sequences. Positions conserving nonpolar residues
(§), polar residues (±), and small residues with near neutral
polarity (") are indicated. Capital letters represent
positions with high conservation and lowercase letters
modest conservation.
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As in GCN2, CPC3 contains a HisRS-like domain located immediately
C-terminal to the kinase domain (amino acids 981-1507, Fig. 5). It is characterized by 34/60%
positional identity/similarity with the HisRS-like domain of GCN2 and
30/52% with DGCN2 (30/53% between GCN2 and DGCN2). All three
HisRS-like domains are characterized by three motifs conserved among
class II aminoacyl-tRNA synthetases, plus sequences unique to HisRS
proteins (58). However, certain residues conserved in genuine HisRS
proteins were absent in CPC3, GCN2, and DGCN2. These include the
invariant Arg in motif 3 that contributes to ATP binding (59), the
amino acid stretch LVRGLDYY (called "histidine A"), and the
(R/K)G-patch N-terminal of motif 1 (62). In contrast, the sequence
AAGGRYD (called "histidine B") is well conserved. Both histidine A
and B motifs were shown to participate in forming the binding pocket
for histidine (60). The Arg residue in histidine A plays a catalytic
role in histidine activation (59, 61, 62), and the (R/K)G-patch is also
vital for full HisRS enzymatic activity (62), but both are missing in
the HisRS-like domains of the eIF2 kinases. The lack of conserved sequences listed above suggested that all three HisRS-like domains lack
the ability to bind histidine and ATP and, thus, should be enzymatically inactive.
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Fig. 5.
Multi-alignment of HisRS-like domains of
CPC3, GCN2, and DGCN2 (explanation see Fig. 4) and genuine HisRS
sequences (Program GCG, gap creation penalty 4, gap extension penalty
2). HisRS sequences were taken from human (hsHisRS,
accession number P12081), yeast (scHisRS, accession number
P07263), and E. coli (ecHisRS, accession number
P04804). Below the alignment, consensus sequences are given for HisRS
positions (c HisRS), for HisRS-like proteins (c
Hlike), and for all proteins (consens). Lowercase
letters indicate moderate conservation (at least 60%) and
capital letters identity in all considered proteins.
Non-equivalent amino acids (#) between consensus sequences of HisRS and
HisRS-like domains are marked. HisRS motifs 1-3 are shown
above the alignment (81), and the respective amino acids are
described as either "small (PGST), § hydrophobic (FYWIMVLA),
+positive (HRK),  negative (DENQ), or * invariant (82). Also the
position and the characteristic sequences are shown from motifs
histidine A and B (60) and from the patches (R/K)G (62) and VAILGE
(62). Positions of mutations in GCN2 mentioned in the text are
underlined: Y1119L, R1120L, A1197G, N1295D, and
H1308Y.
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The most similarity between HisRS-like domains and genuine HisRS is
found in the region between motifs 1 and 3, especially N-terminal to
motif 2. Motif 2 is involved in tRNA binding (63), and the adjacent
N-terminal sequences are uniquely conserved among genuine HisRS
proteins (64, 65). It is not known which amino acids in authentic HisRS
are responsible for the recognition of tRNAHis. However,
GCN2 mutations in the HisRS-like domain are known that lead
to either inactivation (gcn2 alleles) or constitutive
activation (GCN2c alleles) of the kinase domain.
Because of their predicted functional importance, CPC3 was inspected
for the amino acids residing at the following positions: mutation
gcn2-m2 (Y1119L, R1120L) affects amino acids in motif 2 and
was shown to impair in vitro binding of tRNAs to GCN2 (9).
Both residues are conserved in CPC3 and DGCN2. In contrast, some of the
GCN2c mutations affect amino acids that are not
conserved in CPC3 or DGCN2 (e.g.
GCN2c-N1295D and
GCN2c-H1308Y, see Ref. 25) (Fig. 5).
Quite the opposite, the Ala residue altered by the
GCN2c-A1197G mutation (25)
establishes identity with CPC3 at this position. This may indicate that
in these areas the tertiary structure of the protein, not the amino
acids, is conserved.
For the most C-terminal region of CPC3 (amino acids 1507-1646) the
highest similarity found in searching the data bases was with GCN2
(32/58% identity/similarity); however, pairwise comparison of CPC3 and
DGCN2 showed similarities to almost the same degree (27/51%) (28/49%
between GCN2 and DGCN2). No extensive stretches of identical amino
acids could be found in this area which was shown for GCN2 to be
responsible for interaction with ribosomes (25).
In the N-terminal region directly preceding the eIF2 kinase domain
cpc-3 encodes a degenerate kinase domain (amino acids 257-550) also found in GCN2 (8, 66) and DGCN2 (18) (Fig. 1D). The degenerate kinase domains in GCN2 and DGCN2 lack
certain invariant amino acids characteristic of protein kinase
subdomains. In contrast, CPC3 contains all invariant residues; however,
CPC3 lacks the nearly invariant amino acids Phe and Gly in subdomain VII, which participates in ATP binding. It also lacks one nearly invariant amino acid in subdomain I. At those three positions CPC3
contains nonconserved amino acids that are not found in any of the
kinases compared by Hanks and Hunter (12). There are additional
derivations from highly conserved kinase domain sequences that are
unique to each degenerate kinase (for details, see Fig. 3). Two other
unusual features shared by all three degenerate kinase domains are:
first, the presence of an insertion between subdomains I and II;
second, subdomains X and XI are located much closer together than is
observed in genuine kinases. Therefore, we presume that all three
degenerate kinases are catalytically inactive as protein kinases.
However, they may still bind substrates, ATP, and/or regulatory
proteins.
The most N-terminal sequence of CPC3 (amino acids 1-260) showed
significant similarity to GCN2 and DGCN2 (Fig. 3) but to no other
proteins in the data bases available for the on-line BLAST search.
From the extended structural similarities between CPC3, GCN2, and DGCN2
proteins it was concluded that they represent functionally homologous
proteins.
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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Fig. 6.
Construction of the
cpc-3::hph disruption plasmid. The deletion
construct of cpc-3 was engineered with the subcloned
contiguous BamHI restriction fragments ES13 (in plasmid
pES13-1) and ES131 (in plasmid pES131-5) (Fig. 1) as follows.  The
2-kb BamHI-BglII cpc-3 fragment
(black bar) of pES13-1 was cloned blunt-ended into
XhoI-digested pBluescript resulting in plasmid pESI3/8.
BglII digestion resulted in the loss of a 0.6-kb inner
cpc-3 sequence (white bar).  The
Bml cassette (hatched bar) was PCR-amplified from
pBT6 (35) using primers BmlU/BmlL provided 5'
with BglII sites. The BglII-restricted PCR
fragment was cloned into the compatible BamHI site of
pESI3/8 resulting in plasmid pESX14/4. (Function of
BmlR was confirmed by transformation of N. crassa with pES14/4 and detection of benomyl-resistant colonies.)
SalI fragment (cross-hatched bar) from
plasmid pCSN43 containing the hph cassette was ligated into
similarly digested pES131-5 upstream of the N. crassa cpc-3
sequences (flanking the disruption at the 3' side), resulting in
plasmid pESIII2/19. SalI digestion of pES131-5 deleted a
0.4-kb fragment of inner cpc-3 sequence, which together with
the 0.6-kb deletion resulting from step 1 amounted to a 1.0-kb deletion
of the cpc-3 sequence (deleting parts of the eIF2 kinase
and HisRS-like domains).  The XhoI fragment of pESIII2/19
carrying the hph cassette and a 3.5-kb N. crassa
3' sequence was inserted into the compatible SalI site of
pESX14/4 in between the 5' cpc-3 sequence and the
Bml cassette, resulting in disruption plasmids pESXII3/100
and pESXII3/149.
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Fig. 7.
Strategy to disrupt and partially delete the
N. crassa cpc-3 gene. Integration of the plasmid-borne
cpc-3 disruption construct (A) via a homologous
double cross-over event on either side of the hph cassette
should replace the cpc-3 wild-type allele (B) by
the mutant allele cpc-3::hph (C).
Homologous integration should be indicated by 1) stable hygromycin
resistance but 2) benomyl sensitivity and 3) amplification of a 2.1-kb
PCR fragment with primer pair 11.1/11.2 (but no amplification with
primers 2.1/2.3 in homokaryotic
cpc-3::hph strains). 4) In a Southern
analysis of XhoI-digested genomic DNA of the mutant, a 6-kb
fragment (as opposed to a 4-kb fragment in the wild-type) should be
detected using the 6.1-6.2 fragment (Fig. 1B) as probe.
black bar cpc-3 gene, cross-hatched bar hph
cassette, hatched bar Bml cassette.
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The cycloheximide-resistant strain cyh-2 was transformed
with plasmid pESXII3/100 bearing the
cpc-3::hph;Bml disruption construct (Fig. 7). Of
595 hygromycin-resistant transformants that were isolated, 322 were
sensitive to benomyl, and of these 52% carried an unstable hygromycin
resistance and were discarded. PCR analysis of genomic DNA of the
remaining 166 candidates with primer pair 11.1/11.2 yielded the
anticipated 2.1-kb fragment (Fig. 7) from two strains, S1/148 and
S1/152. From both primary transformants 10 potentially homokaryotic
microconidial subcultures where isolated, and their DNA was subjected
to PCR reactions using primer pairs 11.1/11.2 or 2.1/2.3, respectively.
Out of the 10 S1/152-derived cultures 8 allowed amplification of
11.1-11.2 fragments only (not shown) indicating homokaryosity for the
cpc-3::hph allele. Persistent heterozygosity in
all microconidial subcultures from transformant S1/148 was indicative
of a lethal event in the transformed nucleus.
Southern analysis of genomic DNA of S1/148, S1/152, and the
S1/152-derived subcultures was conducted to confirm the results of the
PCR analysis. The data in Fig. 8 verified
that strains S1/152M1-M5 and M7-M9 were homokaryotic for the
cpc-3::hph allele. The primary transformant S1/148
showed correct fragment sizes in the Southern hybridization (Fig. 8),
and no ectopic integration of the transformant plasmid was found which
might have accounted for disruption of an essential gene. The lethal
event could have occurred due to the mutagenic nature of the
transformation procedure itself. Viable hygromycin-resistant segregants
of a cross between S1/148 and wild-type provided evidence that the
postulated lethality was not correlated with the
cpc-3::hph gene disruption. In any event, the
difference between primary transformants S1/148 and S1/152 documented
that two independent transformants with site-specific integration
events were obtained.
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Fig. 8.
Test for homologous integration of the
disruption construct cpc-3::hph at the
cpc-3 locus by Southern analysis. 10 µg of
XhoI-digested DNA of the recipient strain cyh-2,
the primary transformant S1/152, and 10 of its microconidial
subcultures (M1-10), and the primary transformant S1/148
were electrophoretically separated and probed with 6.1-6.2 DNA (Fig.
1B). DNA of microconidial strain S1/152M10 (M10)
showed a signal at 6 kb after longer exposure. S1/152, S1/148, and
S1/152-derived microconidial strains contained a 6-kb signal, expected
after gene replacement; however, the primary transformants and
S1/152-derived subcultures M6 and M10 additionally possessed the native
4-kb fragment present in the cyh-2-recipient strain,
indicative of heterokaryosis. By using all sequences involved in the
disruption plasmid as probe, no further signals were obtained
indicating that no additional ectopic integrations had occurred (not
shown).
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Disruption of the correct gene was supported by linkage studies. In
crosses between homokaryotic derivatives of S1/152 (putative genotype
cpc-3::hph, cyh-2) and wild-type, a total of 58 segregants was tested for hygromycin and cycloheximide resistance. Each
locus segregated in a 1:1 ratio, but only three of the segregants
differed from the parental allele combinations (not shown) in agreement with 4.2% recombination found in the restriction fragment length polymorphism mapping studies between a molecular marker for
cpc-3 and the cyh-2 locus (see above).
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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Fig. 9.
Effect of the cpc-3 disruption on
the derepression of L-ornithine carbamoyltransferase (OTC)
in histidine-deprived mycelia. Specific activity was measured in
the cyh-2 recipient strain, homokaryotic
cpc-3::hph mutants (cpc-3, average of strains
S1/152M1, -2, -3, -4, -5, -7, -8, -9), heterokaryotic
cpc-3+/cpc-3::hph
transformants (cpc-3+/cpc-3, average of strains S1/152M6 and M10),
and heterokaryotic primary transformants S1/148 and S1/152 after
exponential growth on standard medium or 4 h after supplementation
with 6 mM (final) 3AT, respectively. For comparison strains
with mutations at the cpc-1(j-5) and cpc-2(U142)
loci and the wild-type were investigated under the same
conditions.
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To show that the cpc-3 mutation specifically impairs the
expression of amino acid biosynthetic enzymes, an enzyme belonging to
the citric acid cycle, citrate synthetase, was investigated on
different carbon sources. Its regulation was not affected by the
cpc-3 mutation (not shown) demonstrating that the
cpc-3 mutation did not abolish derepression in general.
Since the general amino acid control activates transcription of the
biosynthetic target genes in amino acid-starved cells (23), the
arg-12 transcript was investigated in the homokaryotic cpc-3 isolates by Northern analysis. In contrast to the
increased arg-12 transcript level found in the wild-type
grown on 3AT (Fig. 10A) only
a very low transcript level was observed in cpc-3 mutant strains irrespective of amino acid sufficiency, i.e. the
arg-12 transcript induction appeared completely dependent on
a functional cpc-3 allele. These results allowed us to
conclude that cpc-3, like GCN2, supplies a
positive function critically required for transcriptional derepression
of genes subject to general amino acid control.
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Fig. 10.
Investigation of gene expression in
cpc-3 mutants. Northern analysis was performed with
total cellular RNA of N. crassa grown exponentially on
standard medium (M) or 4 h after supplementation with 6 mM (final) 3AT (A). A, the same
membrane containing RNA from the homokaryotic cpc-3 mutants
S1/152M8 and M9, the wild-type, and a cpc-1(j-5) strain was
probed with [-32P]dCTP-labeled plasmids carrying the
arg-12, cpc-1, cpc-2, or actin genes,
respectively, the latter an internal control for equal loading.
B, quantitative estimation of cpc-1 mRNA
levels of the heterokaryotic strain S1/152M6 (cpc-3/cpc-3+),
cpc-3 mutants (average of S1/152M8 and M9), and wild-type
after supplementation of 3AT. The signal intensities of the hybridizing
probes cpc-1 and actin were calculated using NIH Image, and
the amount of cpc-1 mRNA was normalized to the actin
mRNA level of each sample, respectively. The cpc-1
mRNA level of wild-type was set to 100%. The cpc-1
mRNA levels of the cpc-3 mutants did not vary
significantly (S1/152M8, 58%; M9, 60%), and therefore the average
level is shown including the standard variation.
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In N. crassa amino acid deprivation elicits a strong
increase in the mRNA level of the cpc-1 transcriptional
activator (3, 30, 69). The cpc-3 mutation did not prevent
the substantial up-regulation of cpc-1 transcript level in
response to amino acid deprivation (Fig. 10A), with the
mRNA increased to 58% of the wild-type level (Fig.
10B). Previous investigations of cpc-1 mutants
(29, 34) had shown that derepression of the arg-12
transcript depends completely on a functional cpc-1 gene.
The finding that no derepression of arg-12 mRNA occurred
in the cpc-3 mutant despite a substantial increase in
cpc-1 transcription (see above) is consistent with a
function of cpc-3 in stimulating cpc-1
translation.
With respect to cpc-2 it was found that the cpc-3
mutation abolished the down-regulation of cpc-2 mRNA in
amino acid-deprived cells, as described previously for cpc-1
mutations (31). On the other hand, in cpc-2;cpc-3
and cpc-2;cpc-1 double mutants the
cpc-3 and cpc-1 mutations did not mask the
phenotypes characteristic for a cpc-2 mutation,
i.e. reduced growth rate (50%) and female infertility (data
not shown), thereby indicating a broader function of cpc-2
operating outside the mechanism of general control.
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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Fig. 11.
Effect of cpc-3 mutation on
cellular CPC1 protein level. CPC1 was detected by immunoblot
analysis of crude cell extracts derived from wild-type, and
cpc-1(j-5) and cpc-3 (S1/152M9 once backcrossed
with wild-type) mutants, after exponential growth in minimal medium
(M) or 4 h after supplementation of 6 mM
(final) 3AT (A). Aliquots of each sample containing 50 µg
of protein were separated in an SDS-polyacrylamide gradient gel
(4-12%) and transferred to nitrocellulose membranes (NOVEX). The
anti-CPC1-antiserum from rabbit (49) was not affinity purified prior to
use, in contrast to previously published work (49). The
arrow indicates the position of CPC1. The apparent mass of
CPC1 based on its electrophoretic mobility is higher than predicted
from its nucleotide sequence (30 kDa), even higher than observed
previously (3), which perhaps can be explained by the use of different
gel systems. A large discrepancy between predicted and apparent
molecular mass has been noted also for GCN4, the homologue protein in
yeast (87). The nonspecific signals were used as internal controls for
equal loading of proteins. The position of the molecular mass markers
are indicated by > 98, 64, 50, 36 and 30 kDa.
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DISCUSSION |
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
G1, 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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X91867.
Kinase and Histidyl-tRNA Synthetase-related Domains
Required for General Amino Acid Control*
§¶,
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Abstract
Introduction
