Role of a novel endoplasmic reticulum-resident glycoprotein Mtc6/Ehg2 in high-pressure growth: stability of tryptophan permease Tat2 in Saccharomyces cerevisiae.

Deep-sea organisms are subjected to extreme conditions; therefore, understanding their adaptive strategies is crucial. We utilize Saccharomyces cerevisiae as a model to investigate pressure-dependent protein regulation and piezo-adaptation. Using yeast deletion library analysis, we identified 6 poorly characterized genes that are crucial for high-pressure growth, forming novel functional modules associated with cell growth. In this study, we aimed to unravel the molecular mechanisms of high-pressure adaptation in S. cerevisiae, focusing on the role of MTC6. MTC6, the gene encoding the novel glycoprotein Mtc6/Ehg2, was found to stabilize tryptophan permease Tat2, ensuring efficient tryptophan uptake and growth under high pressure at 25 MPa. The loss of MTC6 led to promoted vacuolar degradation of Tat2, depending on the Rsp5-Bul1 ubiquitin ligase complex. These findings enhance our understanding of deep-sea adaptations and stress biology, with broad implications for biotechnology, environmental microbiology, and evolutionary insights across species.

Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2.

In Saccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 µg/mL). The Km value of wild-type Tat2 for tryptophan appeared to be 24 µg/mL, whereas that of Tat2I285V and Tat2I285T was 17 and 11 µg/mL, respectively. Normalized values of Vmax/Km for Tat2I285V- and Tat2I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3-11.7) of Escherichia coli AdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H+ symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2I285V and Tat2I285T levels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 µg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.

Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae.

Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.

Lipid droplet proteins, Lds1p, Lds2p, and Rrt8p, are implicated in membrane protein transport associated with ergosterol.

Lipid droplets (LDs) are ubiquitous organelles, enclosed in a monolayer of phospholipid, which store excess fatty acids as neutral lipids such as triacylglycerol and sterol esters. Previous studies have revealed that LDs contain many proteins with various functions required for lipid metabolism and vesicular trafficking. Among them, Lds (Lipid Droplet in Sporulation) proteins, Lds1p and Lds2p, are reportedly induced and localized to LDs during yeast sporulation, but their cellular function has not been clarified. Here we show that the Lds proteins, Lds1p, Lds2p and Rrt8p, are expressed and localized at LDs in vegetative cells, being required for proper localization of plasma membrane proteins. We found that deletion of Lds genes led to mis-sorting of Wsc1p, a cell wall stress sensor, from the plasma membrane to the vacuole. We also demonstrated that lack of these proteins partially suppressed the growth defect and mis-sorting of the high-affinity tryptophan transporter Tat2p, induced by impairment of ergosterol biosynthesis. Furthermore, we identified Sec39p/Dsl3p, a component of the DSL1 tethering complex that mediates the interaction with COPI vesicles, as a binding partner for Lds2p. These results suggest a possible role of Lds proteins in maintenance of membrane lipid homeostasis and accompanying membrane protein transport.

Molecular functions of the iron-regulated metastasis suppressor, NDRG1, and its potential as a molecular target for cancer therapy.

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.

Retention of chimeric Tat2-Gap1 permease in the endoplasmic reticulum induces unfolded protein response in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR.

RiTe conjugate mediated corneal collagen crosslinking, a novel therapeutic intervention for keratoconus - in vitro and in vivo study.

Corneal collagen crosslinking (CXL) is an effective method to halt the disease progression of keratoconus, a progressive corneal dystrophy leading to cone shaped cornea. Despite the efficacy of standard protocol, the concerning step of this procedure is epithelial debridement performed to facilitate the entry of riboflavin drug. Riboflavin, a key molecule in CXL protocol, is a sparsely permeable hydrophilic drug in corneal tissues. The present study has employed cell penetrating peptide (CPP), Tat2, to enhance the penetration of riboflavin molecule, and thereby improve currently followed CXL protocol. This study demonstrates approximately two-fold enhanced uptake of CPP riboflavin conjugate, Tat2riboflavin-5'Phosphate (RiTe conjugate), both in vitro and in vivo. Two different CXL protocols (Epi ON and Epi OFF) have been introduced and implemented in rabbit corneas using RiTe conjugate in the present study. The standard and RiTe conjugate mediated CXL procedures exhibited an equivalent extent of crosslinking in both the methods. Reduced keratocyte loss and no endothelial damage in RiTe conjugate mediated CXL further ascertains the safety of the proposed CXL protocols. Therefore, RiTe conjugate mediated CXL protocols present as potential alternatives to the standard keratoconus treatment in providing equally effective, less invasive and patient compliant treatment modality.

Substrate-induced differential degradation and partitioning of the two tryptophan permeases Tat1 and Tat2 into eisosomes in Saccharomyces cerevisiae.

Tryptophan is a relatively rare amino acid whose influx is strictly controlled to meet cellular demands. The yeast Saccharomyces cerevisiae has two tryptophan permeases, namely Tat1 (low-affinity type) and Tat2 (high-affinity type). These permeases are differentially regulated through ubiquitination based on inducible conditions and dependence on arrestin-related trafficking adaptors, although the physiological significance of their degradation remain unclear. Here, we demonstrated that Tat2 was rapidly degraded in an Rsp5-Bul1-dependent manner upon the addition of tryptophan, phenylalanine, or tyrosine, whereas Tat1 was unaffected. The expression of the ubiquitination-deficient variant Tat25K>R led to a reduction in cell yield at 4 µg/mL tryptophan, suggesting the occurrence of an uncontrolled, excessive consumption of tryptophan at low tryptophan concentrations. Eisosomes are membrane furrows that are thought to be storage compartments for some nutrient permeases. Tryptophan addition caused rapid Tat2 dissociation from eisosomes, whereas Tat1 distribution was unaffected. The 5 K > R mutation had no marked effect on Tat2 dissociation, suggesting that dissociation is independent of ubiquitination. Interestingly, the D74R mutation, which was created within the N-terminal acidic patch, stabilized Tat2 while reducing the degree of partitioning into eisosomes. Moreover, the hyperactive I285V mutation in Tat2, which increases Vmax/Km for tryptophan import by 2-fold, reduced the degree of segregation into eisosomes. Our findings illustrate the coordinated activity of Tat1 and Tat2 in the regulation of tryptophan transport at various tryptophan concentrations and suggest the positive role of substrates in inducing a conformational transition in Tat2, resulting in its dissociation from eisosomes and subsequent ubiquitination-dependent degradation.

Enhanced in vivo antifungal activity of novel cell penetrating peptide natamycin conjugate for efficient fungal keratitis management.

Natamycin is the only FDA approved drug that is used as a first line of treatment for fungal keratitis caused by filamentous fungi, however natamycin is known for poor corneal penetration. Cell penetrating peptides (CPPs) are emerging nanocarriers for the enhanced delivery of various macromolecules owing to their distinct cellular translocation ability. In the present study, tissue penetration ability and antifungal efficacy of CPP (Tat2) conjugated natamycin has been investigated and compared with natamycin alone in vivo. Results show that Tat2natamycin exhibits five- fold higher ocular penetration than natamycin alone when given topically. Complete resolution of fungal keratitis in 44% of the animals in Tat2natamycin treated group as compared to only 13% of the animals in natamycin treated group further highlights its increased antifungal efficacy. Hence, this conjugate is a promising antifungal molecule with enhanced ocular penetration as well as antifungal efficacy against selected fungal species.

TORC1 ensures membrane trafficking of Tat2 tryptophan permease via a novel transcriptional activator Vhr2 in budding yeast.

The target of rapamycin complex 1 (TORC1) protein kinase is activated by nutrients and controls nutrient uptake via the membrane trafficking of various nutrient permeases. However, its molecular mechanisms remain elusive. Cholesterol (ergosterol in yeast) in conjunction with sphingolipids forms tight-packing microdomains, "lipid rafts", which are critical for intracellular protein sorting. Here we show that a novel target of rapamycin (TOR)-interacting transcriptional activator Vhr2 is required for full expression of some ERG genes for ergosterol biogenesis and for proper sorting of the tryptophan permease Tat2 in budding yeast. Loss of Vhr2 caused sterol biogenesis disturbance and Tat2 missorting. TORC1 activity maintained VHR2 transcript and protein levels, and total sterol levels. Vhr2 was not involved in regulation of the TORC1-downstream protein kinase Npr1, which regulates Tat2 sorting. This study suggests that TORC1 regulates nutrient uptake via sterol biogenesis.

pH Biosensing by PI4P Regulates Cargo Sorting at the TGN.

Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pKa values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by TGN PI4P in response to nutrient availability governs protein sorting at the TGN, likely by regulating sterol transfer to the TGN by Osh1, a member of the conserved oxysterol-binding protein (OSBP) family of lipid transfer proteins. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.

Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast.

Low-temperature fermentations produce wines with greater aromatic complexity, but the success of these fermentations greatly depends on the adaptation of yeast cells to cold. Tryptophan has been previously reported to be a limiting amino acid during Saccharomyces cerevisiae growth at low temperature. The objective of this study was to determine the influence of the tryptophan metabolism on growth and fermentation performance during low-temperature wine fermentation. To this end, we constructed the deletion mutants of the TRP1 and TAT2 genes in a derivative haploid of a commercial wine strain, and the TAT2 gene was overexpressed in the prototroph and auxotroph (Δtrp1) backgrounds. Then we characterized growth and fermentation activity during wine fermentation at low and optimum temperatures. Our results partially support the role of this amino acid in cold yeast growth. Although deletion of TRP1 impaired amino acid uptake and the growth rate at low temperature in synthetic must, this growth impairment did not affect the fermentation rate. Deletion of TAT2 endorsed this strain with the highest nitrogen consumption capacity and the greatest fermentation activity at low temperature. Our results also evidenced reduced ammonium consumption in all the strains at low temperature.