The GCN4 Transcription Factor: A Review of Its Functional Progress in Fungi

Abstract

Nitrogen serves as a pivotal nutrient for the proliferation, maturation, and pathogenicity of fungi. Despite its importance, nitrogen starvation is a common challenge encountered during fungal development and host invasion. A key regulatory transcription factor, known as general control non-derepressible 4 (GCN4), has been characterized in various fungal groups, including model fungal, pathogens, and basidiomycetes. This factor is triggered by nitrogen limitation and subsequently stimulates the expression of a multitude of genes involved in amino acid synthesis, thereby countering the effects of nitrogen deficiency. This paper provides a comprehensive review on the activation mechanisms, the structural characteristics and stability of GCN4, and how GCN4 activates its downstream target genes to regulate the physiological processes of fungi. This study lays the theoretical groundwork for future research endeavors that seek to enhance nitrogen utilization, preserve the delicate balance of carbon–nitrogen metabolism, and stimulate growth, development, and secondary metabolism in fungi, especially under nitrogen-limited conditions.

1. Introduction

Nutrients participate in biochemical reactions as components that generate energy or contribute to the formation of cellular biomass. Among them, nitrogen sources are one of the fundamental components for synthesizing nucleic acids and proteins, playing a crucial role in basic metabolism, growth, and development. During fungal growth, a phenomenon of nitrogen deficiency often occurs, which affects the growth, development, and secondary metabolism of fungi [1]. In early research on yeast, an important transcription factor, general control non-derepressible 4 (GCN4), was identified, which mediates the general amino acid control (GAAC) process [2,3]. This process is capable of regulating the expression of numerous genes related to amino acid biosynthesis. In organisms such as Aspergillus nidulans [4], A. fumigatus [5], Neurospora crassa [6], and Lentinula edodes, its homologs has been designated as CPC1 or CPCA. GCN4 is a basic leucine zipper (bZIP) transcription factor, belonging to the AP1 (Jun/Fos) subfamily. It exerts regulatory functions by forming a scissor-like spatial structure as a dimer, which enables it to bind to DNA [7,8]. Its unique 5′ untranslated region (5′ UTR) structure endows GCN4 with a distinctive translational regulatory function [9]. Although GCN4 was initially identified as the primary transcription factor controlling intracellular amino acid synthesis, subsequent research have revealed that GCN4 is also involved in the regulation of genes related to antioxidant enzymes, mitochondrial carrier proteins, autophagy, and circadian rhythm genes [10,11,12]. Furthermore, it modulates the growth, development, and secondary metabolism of fungi [5,13,14,15,16].

2. Research Progress on the GCN4-Mediated General Amino Acid Control (GAAC) Signaling Pathway

In Saccharomyces cerevisiae, the deprivation of a single amino acid leads to an increased expression of genes that encode enzymes for multiple amino acid biosynthetic pathways, known as GAAC [2]. The transcription factor GCN4 is a component of the GAAC signaling pathway [3], which can be activated by amino acid starvation, with the GAAC signal being transduced through the GCN2-eIF2α-GCN4 signaling cascade. General control non-derepressible 2 (GCN2) is a serine/threonine protein kinase that is activated in response to uncharged tRNA during amino acid starvation. Once activated, GCN2 phosphorylates the eukaryotic initiation factor 2α(eIF2α), leading to an enhancement in the translation of GCN4 [17]. This, in turn, regulates the transcription of downstream genes involved in amino acid anabolism and other interconnected cellular processes.

2.1. The Translation Mechanism of GCN4

The GCN4 mRNA leader is unusually long (~600 nt) and contains four short upstream open reading frames (uORFs) with only two or three codons each (designated uORF1 to uORF4 from the 5′ to the 3′ end) [18]. In addition, there is a downstream primary protein-coding open reading frame (pORF or mORF). These uORFs enable GCN4 to respond to environmental changes for translational regulation and to restrict the GCN4 protein content under specific stress conditions. Constructing point mutations in the 5′ leader start codon indicates that uORF is crucial for the translational repression of GCN4 [19]. In S. cerevisiae, the interplay between the translation initiation machinery and the 5′ untranslated region (5′ UTR) of GCN4 mRNA plays a crucial role in responding to nutritional stress, particularly amino acid starvation [9].

Under non-starvation conditions (Figure 1A), the eukaryotic initiation factor 2 (eIF2) binds guanosine triphosphate (GTP) and Met-tRNA_i^Met to form a ternary complex (TC). This complex associates with the 40S ribosome and other eIFs to form a 43S preinitiation complex (PIC). Acting under the influence of the eIF4 family of factors, the ribosomal complex binds near the 5′ end of the mRNA and scans the mRNA in a downward direction to locate the AUG start codon, initiating the translation of uORF1. After translating the uORF1-encoded tripeptide, a subset of ribosomes dissociates, a fraction of the 40S ribosomes continues to scan distally along the mRNA, re-engage with abundant TC, and reinitiate translation at downstream uORFs (uORF3 or uORF4). Due to the opposing function of uORF4 relative to uORF1, the uORF4 sequence is rich in GC at the 3′ end, which is not conducive to reinitiation [20], but is conducive to ribosome dissociation. Therefore, following translation from uORF4, it is challenging for the 40S ribosome to reassociate with the TC and initiate translation of the mORF. Under non-amino acid starvation conditions, the flow of ribosomes to the main protein-coding mORF is restricted up to 100-fold due to the impediment of uORFs, thereby repressing the translation of GCN4 [21]. Under the condition of amino acid starvation (Figure 1B), GCN2, the eIF2α kinase phosphorylates eIF2α at Ser 51 [17,22]. Phosphorylated eIF2α enhances its affinity for the GTP-GDP exchange factor (GEF) eIF2B, competitively inhibiting the rate of nucleotide exchange, thereby leading to a reduction in the formation of the TC [21]. The 40S ribosome, after translating uORF1, cannot timely bind with the TC; thus, it can bypass the initiation codon of the more inhibitory 5′ distal uORF and reinitiate at the AUG codon of the mORF. Under the amino acid starvation condition, the content of GCN4 protein increases up to 10-fold compared to non-starvation conditions [23]. A single inhibitory uORF can regulate the translation of GCN4 in response to amino acid starvation conditions in Candida albicans [24]. Mutations in the structural genes for eIF2α and eIF2β of S. cerevisiae disrupts the translational control of GCN4 mRNA [10]. The mutation of the GCN4 translational repressor gene gcd10 in S. cerevisiae reduces the ability of eIF3 to stimulate the binding of the TC with the 40S ribosomal subunit in vivo, mimicking the phosphorylation of eIF2α that allows ribosomes to bypass uORFs and reinitiate at GCN4 [25]. Inserting sequences that have the potential to form secondary structures around uORF4 can eliminate the repression of GCN4 translation under non-starvation conditions, indicating that ribosomes translating GCN4 do so by ignoring the AUG start codon of uORF4 [26]. The regulation of the expression of tRNA_i^Met, eIF-2, and eIF-2B indicates that GCN4 translation is negatively coupled with the levels of the TC [27]. These studies collectively provide a broad experimental support for the model of GCN4 translational control.

In addition to inducing the transcriptional activation of GCN4 by nitrogen starvation, the deficiency of purines [28], glucose scarcity [29], high salt concentrations [30], the alkylating agent methyl methanesulfonate (MMS) [31], and rapamycin [32] also activate the transcription of gcn4. These stimulate the translation of GCN4 through mechanisms that are identical to those of amino acid-deprived cells. The exception is that the glucose-dependent activation of GCN4 is mediated by the Ras/cAMP pathway, which is also responsible for the UV light-dependent activation of GCN4 but is not involved in the amino acid starvation-induced activation of GCN4 [29].

2.2. The Stability of the GCN4 Protein

After the translation of GCN4 mRNA, the content of GCN4 protein increases rapidly, but the GCN4 protein is highly unstable. Although the majority of cellular proteins are metabolically stable, a subset of proteins is rapidly degraded [33]. This subset includes many regulatory proteins, possibly because rapid turnover allows for effective modulation of protein levels in response to changes in their synthesis rates [34]. Amino acid deficiency stabilizes the levels of GCN4 protein, and the degradation of GCN4 in vivo depends on the presence of two specific ubiquitin-conjugating enzymes, Cdc34 and Rad6, which can direct the ubiquitination of GCN4 in vitro [35]. The degradation of GCN4 also requires the combined action of the kinase Pho85 and the SCF^CDC4 (the SCF complex containing Cdc4 as the F-box component) ubiquitin ligase complex [36]. The accumulation of the threonine pathway intermediate β-aspartate semialdehyde (ASA), which is a substrate for homoserine dehydrogenase (Hom6), can also attenuate the GAAC transcriptional response by accelerating the degradation of GCN4 [37]. The degradation mechanism of GCN4 is linked to the translation process, and there is a potential connection between the translation and stability of GCN4 mRNA. Its translation rate varies with the availability of amino acids. Under conditions that favor its translation, the steady-state level of GCN4 mRNA declines, but this is not due to a measurable change in its decay rate. This transient change in stability is the reason for the readjustment of the steady-state level of GCN4 mRNA [38]. Given the paramount importance of protein stability in regulation, it is therefore necessary to enhance the stability of GCN4. Ssb2, a component of the Hsp70 protein family responsible for protein quality control, increases the synthesis and stability of Gcn4 and rescues the defective phenotype of gcn4 mutants [39]. Methionine guides the conserved methyltransferase Ppm1 to stabilizes the levels of GCN4 through the phosphatase PP2A. When methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the equilibrium of GCN4 towards a dephosphorylated and stable state, reducing the phosphorylation of GCN4 and thereby decreasing its ubiquitination and degradation, even though methionine can still increase GCN4 protein levels under conditions of high cellular growth and translation [40]. The stability differences in the GCN4 protein confer its cell-specific activity in differentiated yeast colonies. The cell-specific activity of GCN4 protein is independent of Gcn2p or other translational or transcriptional regulations. Cell-specific proteasomal degradation is a key mechanism that diversifies GCN4 protein function between the upper parts of the colony (U cells) and the middle and lower parts (L cells) [41]. This regulation ensures the specific spatiotemporal activity of the GCN4 protein within the colony.

3. The Characteristics of Conserved Structural Domain in the Transcription Factor GCN4

GCN4 is a class of bZIP transcription factors identified in S. cerevisiae, encoding trans-activators of amino acid biosynthetic genes, which include two acidic activation domains and a C-terminal bZIP domain [7]. The bZIP structural motif comprises two regions involving the basic region and the leucine zipper region (Figure 2A). The basic region, adjacent to the leucine zipper region, is rich in basic amino acids such as arginine and lysine. Five highly conserved residues, asparagine-235, alanine-238, alanine-239, serine-242, and arginine-243, interact with the bases of DNA through hydrogen bonds or hydrophobic interactions, constituting a bilateral DNA-binding motif composed of a high-charge basic region that directly contacts DNA [8]. The leucine zipper region is characterized by the periodic occurrence of leucine residues, which repeat every seventh amino acid. These Leu residues are positioned on one side of the helix in a linear arrangement, appearing once every two helical turns. Two such helical molecules can form a “zipper” through hydrophobic interactions between Leu residues on one side of the α-helix, which then form a dimer through an α-helical coiled-coil connection [42,43,44]. The zipper-like dimers can adopt both parallel and antiparallel conformations, with GCN4 specifically forming dimers in a parallel orientation. Circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) studies have confirmed that these dimers almost exclusively form an α-helical structure, constituting 100% of the helical content [45,46] The dimerization of GCN4 is a key aspect of specific DNA binding, but research has found that the monomers and dimers of GCN4 bind to their target DNA sites at the same rate [47]. In its inactive state, the N-terminus of the leucine zipper is extended, but upon DNA binding (Figure 2B), the N-terminal of the leucine zipper forms a scissor-like structure that fits into the groove of the DNA to be bound. The bZIP protein monomer first binds to DNA, and then the second monomer binds to the DNA [48,49]. To enhance binding stability, the N-terminal residues (aspartic acid and proline) of the GCN4 DNA-binding domain (DPAAL) can stabilize the helical conformation of the GCN4 basic region, reducing the energetic cost of DNA binding [50].

4. The Transcription Factor GCN4 Regulates the Physiological Processes of Fungi by Activating the Expression of Downstream Target Genes

GCN4 is an important global regulatory factor that controls the expression of 546 genes in S. cerevisiae [51]. Using a modified SELEX method known as G-SELEX, which employs genomic DNA fragments rather than the conventional oligonucleotide libraries, the high-affinity binding site for GCN4 in yeast has been identified as RTGACTCAY, where R represents a purine base and Y represents a pyrimidine base [52]. GCN4 can regulate the expression of genes related to amino acid biosynthesis, antioxidant enzymes, mitochondrial carrier proteins, autophagy, and circadian rhythm-associated genes [10,11,12]. Furthermore, GCN4 can modulate the carbon–nitrogen balance in fungi, affecting growth, development, and secondary metabolism [5,13,14,15,16]. As a significant nutrient-sensing regulatory factor, GCN4 is also closely interconnected with other important nutrient-sensing signaling pathways [53,54,55,56].

4.1. The Transcription Factor GCN4 Responds to Nitrogen Starvation and Regulates the Synthesis of Intracellular Amino Acids

The regulation of downstream target genes by GCN4, with the most extensive research conducted on the regulation of genes related to amino acid synthesis [2,3]. The regulatory role of GCN4 in response to amino acid deficiency is well understood in S. cerevisiae. By adding 3-aminotriazole (3-AT) to the culture medium to simulate histidine starvation in cells, GCN4 recognizes the GCRE element of target genes, activates the transcription of amino acid biosynthetic genes, promotes hyphal growth, and coordinates morphological formation and metabolic responses under amino acid starvation conditions [14]. GCN4 can regulate the expression of genes involved in amino acid biosynthesis, amino acid transporters, aminoacyl-tRNA synthetases, and the expression of genes encoding mitochondrial carrier proteins [57]. In S. cerevisiae, all pathways of amino acid biosynthesis contain multiple genes induced by GCN4, with the exception of the cysteine (Cys) pathway. However, for the Cys pathway, genes involved in the biosynthesis of precursor serine and homocysteine are also induced by Gcn4p [10]. Transcriptional profiling analysis indicates that in Aspergillus. oryzae, 67 amino acid biosynthetic genes are targets of CPC1, and comparison with the Gcn4/CaGcn4 datasets from S. cerevisiae and C. albicans reveals a conserved regulatory subset of 32 genes primarily composed of amino acid biosynthetic genes [6]. GCN4 can not only maintain intracellular amino acid homeostasis by enhancing the tricarboxylic acid (TCA) cycle and glycolysis pathways and inhibiting the activity of targets of the rapamycin complex 1 (TORC1), but also enhance the transcriptional regulation of AreA by interacting with SKO1 to regulate nitrogen utilization in Ganoderma lucidum [58,59]. The A. nidulans CPCA serves as a central transcription factor in the cross-pathway regulatory network of amino acid biosynthesis [4], with its regulation of the tryptophan synthase-encoding gene trpB under the control of CPCA [60].

4.2. The Transcription Factor GCN4 Extends Lifespan

The incidence of misfolded and damaged proteins increases in aging cells, and the reduction in mRNA translation and the decrease in protein content can better maintain proteostasis. Interfering with mRNA translation significantly affects lifespan [61], reducing protein synthesis can extend lifespan [62], all of which indicate that proteostasis is related to lifespan. The synthesis and degradation of proteins both influence protein homeostasis. Protein synthesis is associated with translation, while protein degradation encompasses two processes: one is cellular autophagy, and the other is the ubiquitin-proteasome system (UPS). Multi-omics studies on the GCN4-dependent replicative lifespan extension model have indicated that GCN4 acts as a regulator of S. cerevisiae protein turnover [11]. The reduction in protein synthesis during the aging process is partly mediated by the increased phosphorylation of eIF2α dependent on GCN2 and the translation inhibition dependent on mRNA binding protein Ssd1, and these mechanisms possess at least some non-overlapping functions in reducing protein synthesis during senescence [63]. Overexpressing GCN4 is sufficient to promote longevity and reduce protein biosynthesis [63,64]. Moreover, the overexpression of GCN4 is sufficient to extend lifespan in an autophagy-dependent manner without altering global translation, indicating that GCN4-mediated autophagy induction is the ultimate downstream target activated by GCN2 to extend lifespan [14]. Potential genetic activators of autophagy under conditions of increased GCN4 translation are ATG21, COG1, and MEH1 [58]. Specifically, the RNA polymerase II subunit Rpb9 upregulates the transcription of the atg1 expression in a GCN4-mediated manner effectively and selectively [65]. Additionally, an increase in the activity of the UPS has been demonstrated to extend the lifespan of S. cerevisiae [66], and the GCN4 genetically affects the transcription of genes involved in the UPS and autophagy [11]. The lifespan extension of S. cerevisiae strains lacking large ribosomal subunit proteins (RPL) depends on the upregulation of GCN4 [67]. Other aging regulatory factors, such as the tRNA transporter Los1 and the mitochondrial AAA protease gene (Afg3) [68,69], also exert their life-extending effects through GCN4. The inhibitors of cytosolic tRNA synthetase, borrelidin, lead to an increase in uncharged tRNA, which is sensed by Gcn2 to phosphorylate eIF2α, inhibit translation initiation, and upregulate the translation of GCN4, thereby extending the lifespan of S. cerevisiae [70]. These findings suggest that the absence of GCN4 disrupts cellular homeostasis, triggers significant alterations in interrelated intracellular metabolic pathways, and these disruptions have profound metabolic consequences, ultimately leading to a shortened lifespan [71].

4.3. The Transcription Factor GCN4 Regulates Oxidative Stress

Reactive oxygen species (ROS) and the levels of biochemical antioxidants are in balance within the body. Oxidative stress occurs when this critical equilibrium is disrupted due to an excess of ROS, the depletion of antioxidants, or a combination of both [72]. As a bZIP transcription factor, GCN4 has been elucidated in various fungi, including S. cerevisiae, C. albicans, and A. nidulans, as being associated with oxidative stress. Upon exposure to hydrogen peroxides (hydrogen peroxide and cumene hydroperoxide), thiol oxidants (diamide), and heavy metals (cadmium) that induce oxidative stress, GCN4 is activated through a GCN2-eIF2α-GCN4 dependent pathway and is essential for the oxidative stress response in S. cerevisiae [12]. Catalase is present in almost all aerobic organisms, and it plays an important role in protecting cells from oxidative damage by catalyzing the degradation of hydrogen peroxide into water and oxygen [73]. The histone acetyltransferase GCN5 plays a pivotal role in the repositioning and displacement of promoter nucleosomes, thereby enhancing the assembly of the PIC and the transcriptional activation of a plethora of highly expressed genes. This process is essential for the downstream gene binding by the transcriptional activator GCN4 [74,75,76]. In A. nidulans, the bZIP transcription factor CPC1 interacts with the promoter region of the catalase-3 (cat-3) gene, facilitating the recruitment of GCN5. This interaction results in localized histone acetylation, which modulates the transcriptional activation of cat-3 to an appropriate level in response to oxidative stress [77,78]. Beyond the fungi previously discussed, recent research has delineated a connection between nitrogen metabolism and oxidative stress mediated by GCN4 in the medicinal fungus G. lucidum [79] and the pathogenic Cryptococcus neoformans [78], underscoring the conserved role of GCN4 in the fungal response to oxidative challenges.

4.4. Transcription Factor GCN4 Regulates the Pathogenicity of Fungi

Plant pathogenic fungi affect the quality and yield of agricultural products. When pathogenic fungi invade a host, they encounter an environment lacking in nitrogen sources. Silencing CPC1 in the heterokaryotic fungus Verticillium longisporum results in mutants that are highly sensitive to amino acid starvation, and the infected plants showed symptoms such as stunting or early senescence [80]. The CPC system regulates production of the secondary metabolite toxin, sirodesmin PL, in the ascomycete, Leptosphaeria maculans [81]. After mutating the DNA of the cpc-1 in Cryphonectria parasitica, the growth ability of the fungus on host chestnut tissue is reduced, indicating that cpCPC1 is crucial for the chestnut blight fungus to infect chestnuts [82]. GCN4 also plays an essential role in pathogenicity in plant pathogenic fungi such as Fusarium graminearum [83], Fusarium fujikuroi [84], and Alternaria alternata [85]. Additionally, as pathogen of animal, Aspergillus infections cause pulmonary fungal diseases such as aspergillosis. In A. fumigatus, CpcA is crucial for its pathogenicity and virulence [5].

4.5. The Other Regulatory Functions of the Transcription Factor GCN4

Carbon and nitrogen are the most abundant nutritional elements in all biological organisms, and the harmonized regulation of their metabolic pathways is essential for the rapid physiological adaptation to the quality and concentration of available nutrients [86]. The tricarboxylic acid (TCA) cycle, a pivotal carbon metabolic pathway within organisms, features the intermediate RTG3 in S. cerevisiae, which is transcriptionally activated by GCN4 [21]. In G. lucidum, GCN4 facilitates the expression of GlMPC, thereby participating in the modulation of the TCA cycle and the biosynthesis of ganoderic acid (GA) under nitrogen-limited conditions [79]. In S. cerevisiae, GCN4 modulates triacylglycerol metabolism by regulating the expression of phm8 [87], thereby influencing carbon metabolism. Additionally, GCN4 is activated in response to glucose through the Ras/cAMP signaling pathway, which coordinates the metabolism of glucose with the biosynthesis of amino acids and purines [21]. This regulatory role of GCN4 underscores its multifaceted influence on the metabolic pathways that govern energy and nutrient homeostasis within the S. cerevisiae. In G. lucidum, GCN4 also interacts directly with the reactive oxygen species (ROS) signaling pathway, exerting a negative regulatory effect on GA biosynthesis under nitrogen-limited conditions [79]. These findings collectively demonstrate the role of GCN4 in sustaining the balance of carbon and nitrogen metabolism in fungi, ensuring the availability of energy and the homeostatic equilibrium of essential components necessary for the perpetuation of the cell cycle.

GCN4 has been demonstrated in numerous studies to play an essential role in the growth, development, secondary metabolism, and pathogenicity of fungi. In A. nidulans, studies on extracts from different asexual developmental stages have shown that CPC1 is abundant post-germination and during the early stages of hyphal growth [13]. In C. albicans, amino acid starvation promotes the filamentous growth of C. albicans, and this morphogenetic response is dependent on CaGCN4 [14]. In Pseudomonas oxalaticus, CpcA promotes hyphal growth and the production of extracellular enzymes such as cellulase under non-amino acid starvation conditions [15], and it also negatively regulates penicillin biosynthesis by promoting lysine biosynthesis [16].

Circadian clocks drive molecular and behavioral rhythms that approximate the 24 h cyclicity of our environment. N. crassa has one of the most thoroughly studied circadian rhythm systems and light signaling pathways. [88]. Circadian-controlled eIF2α phosphorylation in A. nidulans is necessary for rhythmic translation initiation [89]. Under amino acid starvation, the kinase GCN2 phosphorylates eIF2α, activating its downstream transcription factor CPC-1. The activated CPC-1 recruits the histone acetyltransferase GCN-5 to establish the appropriate chromatin state at the circadian gene (frq) promoter and activates the rhythmic expression of metabolic genes. This indicates the important role of the GCN2-eIF2α-GCN4 signaling pathway in maintaining robust circadian clock function in response to amino acid starvation [90].

5. The Research Progress on the Connection Between the Transcription Factor GCN4 and Other Signaling Pathways

There has been some progress in studying the direct interactions between GCN4 and other signaling pathways. Current research mainly reports connections between GCN4 and the target of rapamycin complex (TOR) pathway [21], nitrogen catabolite repression (NCR) pathway [55], and the mitogen-activated protein kinase (MAPK) pathway [56].

The highly conserved Rapamycin Target (TOR) kinase is a central regulator of cell growth and metabolism, responding to the availability of nutrients [91]. TOR kinase assembles into two complexes: the TOR Complex 1 (TORC1) and the TOR Complex 2 (TORC2). The GCN4-activated GAAC pathway is closely intertwined with the TOR pathway. The TORC1 pathway is activated by sensing high intracellular amino acid concentrations and is involved in the regulation of protein synthesis [92]. When intracellular amino acids are abundant, the Rag GTPases heterodimer facilitates the localization of TORC1 to the lysosome, where TORC1 binds to Rheb and phosphorylates downstream substrates p70S6K and 4E-BP1, thereby promoting mRNA translation [92,93]. In conditions of amino acid scarcity, amino acid sensors reduce TORC1 activity to mitigate amino acid consumption [92,93]. However, GCN2 is activated in response to the scarcity of intracellular amino acids. Consequently, TORC1 and GCN2 exert regulatory functions by sensing different intracellular amino acid concentration changes. Research on the relationship between TORC1 and GCN2 has shown that under conditions of sufficient nitrogen sources, TORC1 inhibits the activity of GCN2 by phosphorylating its Ser577 residue [94]. However, when TORC1 is inhibited by rapamycin, this leads to the activation of the TORC1 downstream target protein SIT4, which in turn activates GCN2 by dephosphorylating its Ser577 [94]. Additionally, studies have found that under amino acid starvation conditions, GCN2 also directly phosphorylates the regulatory subunit KOG1 of TORC1, resulting in the inhibition of TORC1 activity [51]. In the process of autophagy, TORC1 and TORC2 play opposing roles; TORC1 is a well-recognized negative regulator of autophagy, while TORC2 can act as a positive regulator of autophagy by promoting the activation of the general amino acid control response through its downstream target kinase Ypk1 during amino acid starvation [54].

The general amino acid control (GAAC) pathway, in conjunction with the nitrogen catabolite repression (NCR) pathway, collectively regulates the utilization of nitrogen sources. Under conditions where non-preferred nitrogen sources are present, the cell elevates the transcriptional and translational levels of GCN4, and, subsequently, GCN4 enhances the transcriptional regulation of target genes involved in both the NCR and GAAC pathways [55]. Beyond this, the NCR and GAAC pathways are also capable of preferentially modulating the synthesis of proteins involved in promoting the absorption and synthesis of amino acids, optimizing the catabolism and utilization of non-preferred amino acids. However, the precise collaborative mechanisms underlying this regulation remain to be elucidated [95]. Additionally, research has demonstrated that under specific physiological conditions, the core transcription factors GLN3 and GCN4 within the NCR pathway act synergistically to upregulate a suite of genes. For instance, in S. cerevisiae under purine starvation conditions, GLN3 and GCN4 jointly regulate the expression of the glutamine synthetase gene [96]. Under culture conditions with ethanol addition, they cooperatively regulate the transcription of the NADP-dependent glutamate dehydrogenase gene [97]. Under low nitrogen or amino acid-deficient culture conditions, they collectively enhance the expression of genes related to γ-aminobutyrate (GABA) synthesis, such as UGA3, and the glutamate synthase gene [98,99]. Furthermore, GLN3 and GCN4 have also been reported to co-regulate the transcription of genes involved in uric acid catabolism and the NADP-dependent glutamate dehydrogenase genes. The GAAC pathway and 14-3-3 proteins Bmh1/2 are required for the sensitive regulation of the localization of the two transcriptional activators Gln3 and Gat1, which are subject to nitrogen catabolite repression [100]. This further substantiates the intimate connection between GCN4 and the NCR pathway.

GCN4 has also been reported to have significant connections with the mitogen-activated protein kinase (MAPK) pathway [56]. Current research in this area is predominantly focused on animal cells, with numerous gaps remaining in our understanding of the interrelationships between GCN4 and other signaling or physiological metabolic pathways in S. cerevisiae or other filamentous fungi. Only in S. cerevisiae has it been reported that there exists a positive regulatory loop between the osmotic response MAPK Hog1 and the Gcn2 protein kinase, which contributes to enhancing the cell’s sensitivity to osmotic stress [100].

6. Conclusions and Prospects

GCN4 was initially characterized for its role in responding to amino acid starvation in S. cerevisiae, and this function has been confirmed in filamentous fungi, such as N. crassa, G. lucidum and A. nidulans. Subsequent research has expanded our understanding of GCN4 activation to include conditions beyond amino acid deficiency, such as purine scarcity, glucose depletion, elevated salt concentrations, exposure to the alkylating agent methyl methanesulfonate, and treatment with rapamycin. Transcriptomic analyses have revealed that GCN4 orchestrates the expression of a diverse array of genes, encompassing those involved in amino acid anabolism, vitamin biosynthesis, antioxidant and redox mechanisms, and mitochondrial carrier proteins. Based on research related to GCN4, we have formulated the following model (Figure 3).

In recent years, there has been a surge in research on the induction of autophagy by GCN4 in S. cerevisiae, and, more recently, GCN4 has been implicated in the regulation of circadian rhythms. Additionally, GCN4 has been proven to be crucial for the virulence of pathogenic fungi such as A. fumigatus, C. albicans, and C. neoformans. Investigating autophagy holds profound implications for comprehending and addressing age-related ailments. Meanwhile, the examination of circadian rhythms is crucial for managing the metabolic oscillations that occur during the nutritional constraints imposed by dietary limitations in mammals. Furthermore, delving into the realm of pathogenic fungi can uncover novel therapeutic targets, paving the way for more effective treatments of related diseases. Research on plant pathogenic fungi helps develop effective disease management strategies, reduce losses in agricultural production, and improve crop yield and quality. This indicates that the study of GCN4 is not only in basic research but also approaches human health and agricultural production. Moreover, considering the essential nature of carbon–nitrogen metabolism in both fungi and animals, the regulatory function of GCN4 in sustaining metabolic balance is increasingly capturing scientific interest. However, the underlying mechanisms require a more in-depth exploration to fully elucidate the regulatory nuances of GCN4 in carbon–nitrogen homeostasis.