Overlapping Roles of Yeast Transporters Aqr1, Qdr2, and Qdr3 in Amino Acid Excretion and Cross-Feeding of Lactic Acid Bacteria

Microbial species occupying the same ecological niche or codeveloping during a fermentation process can exchange metabolites and mutualistically influence each other’s metabolic states. For instance, yeast can excrete amino acids, thereby cross-feeding lactic acid bacteria unable to grow without an external amino acid supply. The yeast membrane transporters involved in amino acid excretion remain poorly known. Using a yeast mutant overproducing and excreting threonine (Thr) and its precursor homoserine (Hom), we show that excretion of both amino acids involves the Aqr1, Qdr2, and Qdr3 proteins of the Drug H+-Antiporter Family (DHA1) family. We further investigated Aqr1 as a representative of these closely related amino acid exporters. In particular, structural modeling and molecular docking coupled to mutagenesis experiments and excretion assays enabled us to identify residues in the Aqr1 substrate-binding pocket that are crucial for Thr and/or Hom export. We then co-cultivated yeast and Lactobacillus fermentum in an amino-acid-free medium and found a yeast mutant lacking Aqr1, Qdr2, and Qdr3 to display a reduced ability to sustain the growth of this lactic acid bacterium, a phenotype not observed with strains lacking only one of these transporters. This study highlights the importance of yeast DHA1 transporters in amino acid excretion and mutualistic interaction with lactic acid bacteria.

uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that non-lethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

A Yeast-Based Screening Unravels Potential Therapeutic Molecules for Mitochondrial Diseases Associated with Dominant ANT1 Mutations

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

Targeting Copper Homeostasis Improves Functioning of vps13Δ Yeast Mutant Cells, a Model of VPS13-Related Diseases

Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson’s diseases, respectively, and iron imbalance is observed in Parkinson’s and Alzheimer’s diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A-D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.

RNA-cDNA hybrids mediate transposition via different mechanisms

Retrotransposons can represent half of eukaryotic genomes. Retrotransposon dysregulation destabilizes genomes and has been linked to various human diseases. Emerging regulators of retromobility include RNA–DNA hybrid-containing structures known as R-loops. Accumulation of these structures at the transposons of yeast 1 (Ty1) elements has been shown to increase Ty1 retromobility through an unknown mechanism. Here, via a targeted genetic screen, we identified the rnh1Δ rad27Δ yeast mutant, which lacked both the Ty1 inhibitor Rad27 and the RNA–DNA hybrid suppressor Rnh1. The mutant exhibited elevated levels of Ty1 cDNA-associated RNA–DNA hybrids that promoted Ty1 mobility. Moreover, in this rnh1Δ rad27Δ mutant, but not in the double RNase H mutant rnh1Δ rnh201Δ, RNA–DNA hybrids preferentially existed as duplex nucleic acid structures and increased Ty1 mobility in a Rad52-dependent manner. The data indicate that in cells lacking RNA–DNA hybrid and Ty1 repressors, elevated levels of RNA-cDNA hybrids, which are associated with duplex nucleic acid structures, boost Ty1 mobility via a Rad52-dependent mechanism. In contrast, in cells lacking RNA–DNA hybrid repressors alone, elevated levels of RNA-cDNA hybrids, which are associated with triplex nucleic acid structures, boost Ty1 mobility via a Rad52-independent process. We propose that duplex and triplex RNA–DNA hybrids promote transposon mobility via Rad52-dependent or -independent mechanisms.

The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.