PQ-loop Protein Research Articles

The role of PQL genes in response to salinity tolerance in Arabidopsis and barley.

While soil salinity is a global problem, how salt enters plant root cells from the soil solution remains underexplored. Non‐selective cation channels (NSCCs) are suggested to be the major pathway for the entry of sodium ions (Na+), yet their genetic constituents remain unknown. Yeast PQ loop (PQL) proteins were previously proposed to encode NSCCs, but the role of PQLs in plants is unknown. The hypothesis tested in this research is that PQL proteins constitute NSCCs mediating some of the Na+ influx into the root, contributing to ion accumulation and the inhibition of growth in saline conditions. We identified plant PQL homologues, and studied the role of one clade of PQL genes in Arabidopsis and barley. Using heterologous expression of AtPQL1a and HvPQL1 in HEK293 cells allowed us to resolve sizable inwardly directed currents permeable to monovalent cations such as Na+, K+, or Li+ upon membrane hyperpolarization. We observed that GFP‐tagged PQL proteins localized to intracellular membrane structures, both when transiently over‐expressed in tobacco leaf epidermis and in stable Arabidopsis transformants. Expression of AtPQL1a, AtPQL1b, and AtPQL1c was increased by salt stress in the shoot tissue compared to non‐stressed plants. Mutant lines with altered expression of AtPQL1a, AtPQL1b, and AtPQL1c developed larger rosettes in saline conditions, while altered levels of AtPQL1a severely reduced development of lateral roots in all conditions. This study provides the first step toward understanding the function of PQL proteins in plants and the role of NSCC in salinity tolerance.

Stm1 is a vacuolar PQ-loop protein involved in the transport of basic amino acids in Schizosaccharomyces pombe

The stm1+ (SPAC17C9.10) gene of Schizosaccharomyces pombe is closely related to genes encoding vacuolar PQ-loop proteins, Ypq1, Ypq2, and Ypq3, of Saccharomyces cerevisiae. When stm1+ fused with GFP was expressed in fission or budding yeast, Stm1-GFP localized at the vacuolar membrane. Isolated vacuolar membrane vesicles from S. cerevisiae cells overexpressing stm1+ exhibited stm1+-dependent arginine and lysine uptake activity. Exchange activity of arginine and histidine/arginine, as observed for Ypq2 of S. cerevisiae, was also detected in the vesicles expressing stm1+. The expression levels of stm1+ in S. pombe cells significantly affected the vacuolar contents of lysine, histidine, and arginine. These results suggest that Stm1 is a vacuolar PQ-loop protein involved in the transport of basic amino acids across the vacuolar membrane.

A PQ-loop protein Ypq2 is involved in the exchange of arginine and histidine across the vacuolar membrane of Saccharomyces cerevisiae

In nutrient-rich conditions, basic amino acids are actively accumulated into the vacuoles by H+-coupled transporters in Saccharomyces cerevisiae. In addition to the H+-coupled systems, the existence of an exchanger for arginine and histidine was indicated by kinetic analysis using isolated vacuolar membrane vesicles; however, the gene(s) involved in the activity has not been identified. Here, we show that the uptake activity of arginine driven by an artificially imposed histidine gradient decreased significantly by the disruption of the gene encoding vacuolar PQ-loop protein Ypq2, but not by those of Ypq1 and Ypq3. The exchange activity was restored by the expression of YPQ2. Furthermore, the substitution of a conserved proline residue, Pro29, in Ypq2 greatly decreased the exchange activity. These results suggest that Ypq2 is responsible for the exchange activity of arginine and histidine across the vacuolar membrane, and the conserved proline residue in the PQ-loop motif is required for the activity.

The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry

Membrane asymmetry is a key organizational feature of the plasma membrane. Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that establish membrane asymmetry by translocating phospholipids, such as phosphatidylserine (PS) and phospatidylethanolamine, from the exofacial leaflet to the cytosolic leaflet. Saccharomyces cerevisiae expresses five P4-ATPases: Drs2, Neo1, Dnf1, Dnf2, and Dnf3. The inactivation of Neo1 is lethal, suggesting Neo1 mediates an essential function not exerted by the other P4-ATPases. However, the disruption of ANY1, which encodes a PQ-loop membrane protein, allows the growth of neo1Δ and reveals functional redundancy between Golgi-localized Neo1 and Drs2. Here we show Drs2 PS flippase activity is required to support neo1Δ any1Δ viability. Additionally, a Dnf1 variant with enhanced PS flipping ability can replace Drs2 and Neo1 function in any1Δ cells. any1Δ also suppresses drs2Δ growth defects but not the loss of membrane asymmetry. Any1 overexpression perturbs the growth of cells but does not disrupt membrane asymmetry. Any1 coimmunoprecipitates with Neo1, an association prevented by the Any1-inactivating mutation D84G. These results indicate a critical role for PS flippase activity in Golgi membranes to sustain viability and suggests Any1 regulates Golgi membrane remodeling through protein-protein interactions rather than a previously proposed scramblase activity.

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.

Loss of ATP-dependent lysine uptake in the vacuolar membrane vesicles of Saccharomyces cerevisiae ypq1∆ mutant

Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1∆ mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.

Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin

Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.