The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast

The sla1+ gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4+ inhibits leucine uptake and growth of sla1Δ cells. Here, transcriptome analysis reveals that genes upregulated in sla1Δ cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4+ media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4+-effects, we isolated sla1Δ spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4+-sensitive leucine uptake linked to TOR. We show that 3H-leucine uptake by SSR-any1-F32V cells in NH4+-media is more robust than by sla1Δ cells. Moreover, F32V may alter any1+ function in sla1Δ vs. sla1+ cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.

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Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences

PLoS Genetics ◽

10.1371/journal.pgen.1008893 ◽

2020 ◽

Vol 16 (8) ◽

pp. e1008893

Author(s):

Thareendra De Zoysa ◽

Eric M. Phizicky

Keyword(s):

Amino Acid ◽

General Amino Acid Control ◽

Acid Control ◽

Control Response

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Non-optimal codon usage is critical for protein structure and function of the master general amino acid control regulator CPC-1

10.1101/2020.09.11.294470 ◽

2020 ◽

Author(s):

Xueliang Lyu ◽

Yi Liu

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Synonymous Codon ◽

Critical Role ◽

Amino Acid Starvation ◽

General Amino Acid Control ◽

Acid Control ◽

Control Response ◽

And Function

ABSTRACTUnder amino acid starvation condition, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control-1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here we show that Neurospora cpc-1 has a non-optimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activating the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulating of CPC-1 expression and function, and establish a genetic example of the importance of codon usage in protein structure.Abstract importanceGeneral amino acid control response is critical for organisms to adapt to amino acid starvation condition. The preference to use certain synonymous codons are a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we show that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, altered protein degradation rate and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulating of CPC-1 expression and function, and establish a genetic example of the importance of codon usage in protein structure.

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Nonoptimal Codon Usage Is Critical for Protein Structure and Function of the Master General Amino Acid Control Regulator CPC-1

mBio ◽

10.1128/mbio.02605-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Xueliang Lyu ◽

Yi Liu

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Synonymous Codon ◽

Critical Role ◽

Amino Acid Starvation ◽

General Amino Acid Control ◽

Acid Control ◽

Control Response ◽

And Function

ABSTRACT Under amino acid starvation conditions, eukaryotic organisms activate a general amino acid control response. In Neurospora crassa, Cross Pathway Control Protein 1 (CPC-1), the ortholog of the Saccharomyces cerevisiae bZIP transcription factor GCN4, functions as the master regulator of the general amino acid control response. Codon usage biases are a universal feature of eukaryotic genomes and are critical for regulation of gene expression. Although codon usage has also been implicated in the regulation of protein structure and function, genetic evidence supporting this conclusion is very limited. Here, we show that Neurospora cpc-1 has a nonoptimal NNU-rich codon usage profile that contrasts with the strong NNC codon preference in the genome. Although substitution of the cpc-1 NNU codons with synonymous NNC codons elevated CPC-1 expression in Neurospora, it altered the CPC-1 degradation rate and abolished its amino acid starvation-induced protein stabilization. The codon-manipulated CPC-1 protein also exhibited different sensitivity to limited protease digestion. Furthermore, CPC-1 functions in rescuing the cell growth of the cpc-1 deletion mutant and activation of the expression of its target genes were impaired by the synonymous codon changes. Together, these results reveal the critical role of codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein folding. IMPORTANCE The general amino acid control response is critical for adaptation of organisms to amino acid starvation conditions. The preference to use certain synonymous codons is a universal feature of all genomes. Synonymous codon changes were previously thought to be silent mutations. In this study, we showed that the Neurospora cpc-1 gene has an unusual codon usage profile compared to other genes in the genome. We found that codon optimization of the cpc-1 gene without changing its amino acid sequence resulted in elevated CPC-1 expression, an altered protein degradation rate, and impaired protein functions due to changes in protein structure. Together, these results reveal the critical role of synonymous codon usage in regulation of CPC-1 expression and function and establish a genetic example of the importance of codon usage in protein structure.

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Insulin Signaling and the General Amino Acid Control Response

Journal of Biological Chemistry ◽

10.1074/jbc.m801331200 ◽

2008 ◽

Vol 283 (28) ◽

pp. 19229-19234 ◽

Cited By ~ 28

Author(s):

Sharon E. Malmberg ◽

Christopher M. Adams

Keyword(s):

Amino Acid ◽

Insulin Signaling ◽

General Amino Acid Control ◽

Acid Control ◽

Control Response

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Receptor for Activated C-Kinase (RACK1) Homolog Cpc2 Facilitates the General Amino Acid Control Response through Gcn2 Kinase in Fission Yeast

Journal of Biological Chemistry ◽

10.1074/jbc.m112.445270 ◽

2013 ◽

Vol 288 (26) ◽

pp. 19260-19268 ◽

Cited By ~ 9

Author(s):

Yusuke Tarumoto ◽

Junko Kanoh ◽

Fuyuki Ishikawa

Keyword(s):

Amino Acid ◽

Fission Yeast ◽

General Amino Acid Control ◽

Acid Control ◽

Control Response

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gcd12 mutations are gcn3-dependent alleles of GCD2, a negative regulator of GCN4 in the general amino acid control of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/122.3.543 ◽

1989 ◽

Vol 122 (3) ◽

pp. 543-550 ◽

Cited By ~ 2

Author(s):

C J Paddon ◽

A G Hinnebusch

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Negative Regulator ◽

Regulatory Function ◽

Growth Defect ◽

Gene Products ◽

Wild Type ◽

General Amino Acid Control ◽

Translational Repressor ◽

Acid Control

Abstract GCD12 encodes a translational repressor of the GCN4 protein, a transcriptional activator of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. gcd12 mutations override the requirement for the GCN2 and GCN3 gene products for derepression of GCN4 expression, suggesting that GCN2 and GCN3 function indirectly as positive regulators by negative regulation of GCD12. In addition to their regulatory phenotype, gcd12 mutants are temperature-sensitive for growth (Tsm-) and, as shown here, deletion of the GCD12 gene is unconditionally lethal. Both the regulatory and the Tsm- phenotypes associated with gcd12 point mutations are completely overcome by wild-type GCN3, implying that GCN3 can promote or partially substitute for the functions of GCD12 in normal growth conditions even though it antagonizes GCD12 regulatory function in starvation conditions. The GCD12 gene has been cloned and mapped to the right arm of chromosome VII, very close to the map position reported for GCD2. We demonstrate that GCD12 and GCD2 are the same genes; however, unlike gcd12 mutations, the growth defect and constitutive derepression phenotypes associated with the gcd2-1 mutation are expressed in the presence of the wild-type GCN3 gene. These findings can be explained by either of two alternative hypotheses: (1) gcd12 mutations affect a domain of the GCD2 protein that directly interacts with GCN3, and complex formation stabilizes mutant gcd12 (but not gcd2-1) gene products; (2) gcd12 mutations selectively impair one function of GCD2 that is replaceable by GCN3, whereas gcd2-1 inactivates a different GCD2 function for which GCN3 cannot substitute. Both models imply a close interaction between these two positive and negative regulators in general amino acid control.

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