Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne.

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3ß,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.

Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol.

Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.

Plasma membrane phosphatidylinositol (4,5)-bisphosphate is critical for determination of epithelial characteristics.

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.

Characterization of micron-scale protein-depleted plasma membrane domains in phosphatidylserine-deficient yeast cells.

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane.

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.

An optogenetic system to control membrane phospholipid asymmetry through flippase activation in budding yeast.

Lipid asymmetry in biological membranes is essential for various cell functions, such as cell polarity, cytokinesis, and apoptosis. P4-ATPases (flippases) are involved in the generation of such asymmetry. In Saccharomyces cerevisiae, the protein kinases Fpk1p/Fpk2p activate the P4-ATPases Dnf1p/Dnf2p by phosphorylation. Previously, we have shown that a blue-light-dependent protein kinase, phototropin from Chlamydomonas reinhardtii (CrPHOT), complements defects in an fpk1Δ fpk2Δ mutant. Herein, we investigated whether CrPHOT optically regulates P4-ATPase activity. First, we demonstrated that the translocation of NBD-labelled phospholipids to the cytoplasmic leaflet via P4-ATPases was promoted by blue-light irradiation in fpk1Δ fpk2Δ cells with CrPHOT. In addition, blue light completely suppressed the defects in membrane functions (such as endocytic recycling, actin depolarization, and apical-isotropic growth switching) caused by fpk1Δ fpk2Δ mutations. All responses required the kinase activity of CrPHOT. Hence, these results indicate the utility of CrPHOT as a powerful and first tool for optogenetic manipulation of P4-ATPase activity.

A complex genetic interaction implicates that phospholipid asymmetry and phosphate homeostasis regulate Golgi functions.

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.

Intra-dimer row and inter-dimer row coupling of the vibrational modes of chemisorbed CO on Si(001)-c(4×2) observed by angle-dependent transmission infrared spectroscopy.

The vibrational modes of chemisorbed CO on a Si(001) surface are investigated by means of transmission Fourier-transform infrared absorption spectroscopy. We observed the three components corresponding to the stretching vibration of the terminal-site CO adsorbed on the down-dimer sites of a Si(001) surface. The symmetric stretching vibration and asymmetric stretching vibration are observed separately. This assignment is consistent with the polarization dependence of the incident light and with the vibrational modes obtained by performing theoretical calculations. We found that both the intradimer row and interdimer row coupling should be considered to explain the appearance of the three components.

Generation of 1,25-dihydroxyvitamin D3 in Cyp27b1 knockout mice by treatment with 25-hydroxyvitamin D3 rescued their rachitic phenotypes.

We have reported that 25-hydroxyvitamin D3 [25(OH)D3] binds to vitamin D receptor and exhibits several biological functions directly in vitro. To evaluate the direct effect of 25(OH)D3 in vivo, we used Cyp27b1 knockout (KO) mice, which had no detectable plasma 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] when fed a diet containing normal Ca and vitamin D. Daily treatment with 25(OH)D3 at 250 µg kg-1 day-1 rescued rachitic phenotypes in the Cyp27b1 KO mice. Bone mineral density, female sexual cycles, and plasma levels of Ca, P, and PTH were all normalized following 25(OH)D3 administration. An elevated Cyp24a1 mRNA expression was observed in the kidneys, and plasma concentrations of Cyp24a1-dependent metabolites of 25(OH)D3 were increased. To our surprise, 1,25(OH)2D3 was detected at a normal level in the plasma of Cyp27b1 KO mice. The F1 to F4 generations of Cyp27b1 KO mice fed 25(OH)D3 showed normal growth, normal plasma levels of Ca, P, and parathyroid hormone, and normal bone mineral density. The curative effect of 25(OH)D3 was considered to depend on the de novo synthesis of 1,25(OH)2D3 in the Cyp27b1 KO mice. This suggests that another enzyme than Cyp27b1 is present for the 1,25(OH)2D3 synthesis. Interestingly, the liver mitochondrial fraction prepared from Cyp27b1 KO mice converted 25(OH)D3 to 1,25(OH)2D3. The most probable candidate is Cyp27a1. Our findings suggest that 25(OH)D3 may be useful for the treatment and prevention of osteoporosis for patients with chronic kidney disease.

Phospholipid flippases and Sfk1p, a novel regulator of phospholipid asymmetry, contribute to low permeability of the plasma membrane.

Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells. Consistent with this, lem3Δ sfk1Δ double mutant cells exposed more PS/PE than the lem3Δ mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol-4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3Δ was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3Δ sfk1Δ double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3Δ sfk1Δ mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.

Synthesis of omega-3 long-chain polyunsaturated fatty acid-rich triacylglycerols in an endemic goby, Gymnogobius isaza, from Lake Biwa, Japan.

It is commonly observed that freshwater fish contain lower amounts of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), than marine fish species. In this study, we performed a detailed comparative analysis of phospholipids (PLs) and triacylglycerols (TAGs) from Gymnogobius isaza, a freshwater goby endemic to Lake Biwa inhabiting the lake bottom, and Gymnogobius urotaenia, a related goby that inhabits the shore of Lake Biwa. We found that tissues from G. isaza contain remarkably high amounts of omega-3 LC-PUFAs in both PLs and TAGs. Mass spectrometry analysis of TAGs demonstrated that the most abundant TAG molecular species were TAG (16:0/18:1/20:5), followed by TAG (14:0/18:1/20:5), in which EPA is incorporated into TAG at either the sn-1 or sn-3 positions. We isolated cDNAs encoding acyl-CoA: diacylglycerol acyltransferase designated as GiDGAT1 and GiDGAT2, from G. isaza. Expression studies using a neutral lipid-deficient Saccharomyces cerevisiae mutant strain demonstrated that both GiDGAT1 and GiDGAT2 possessed diacylglycerol acyltransferase activity, and preferential incorporation of LC-PUFA into TAG was observed in the presence of GiDGAT1. This study revealed the novel lipid profiles of G. isaza and identified the enzymes that were involved in the production of PUFA-containing TAGs.

Electronic structure of α-sexithiophene ultrathin films grown on.

We investigated the electronic states of α-sexithiophene (α-6T) on by means of angle-resolved photoelectron spectroscopy using synchrotron radiation. The characteristic features of π states are observed in the valence region. The increase in the population of the S1 band, assigned to the surface state of , upon deposition of α-6T was measured and the change in the electron density was evaluated. The band diagram was constructed based on the measurement of the HOMO level and work function. The work function was found to change with the α-6T thickness in a characteristic manner. We constructed a model of the electron transfer at each growth stage based on the core levels of the substrate (Si 2p, Ag 3d) and α-6T molecule (C 1s, S 2p), as well as the valence state and work function change.

Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast.

Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the cdc50Δ mutant. We identified a mutation of YMR010W encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene CFS1 (cdc fifty suppressor 1). GFP-tagged Cfs1p was partially colocalized with Drs2p and Neo1p to endosomal/late Golgi membranes. Interestingly, the cfs1Δ mutation suppressed growth defects in all flippase mutants. Accordingly, defects in membrane trafficking in the flippase mutants were also suppressed. These results suggest that Cfs1p and flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the cfs1Δ mutation changed PE asymmetry in the plasma membrane. Cfs1p may thus be a novel regulator of phospholipid asymmetry.

Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid flippases in Saccharomyces cerevisiae.

In eukaryotic cells, phosphatidylserine (PS) is predominantly located in the cytosolic leaflet of the plasma membrane; this asymmetry is generated by an unknown mechanism. In this study, we used the PS-specific probe mRFP-Lact-C2 to investigate the possible involvement of type 4 P-type ATPases, also called phospholipid flippases, in the generation of this asymmetry in Saccharomyces cerevisiae. PS was not found in the trans-Golgi Network in wild-type cells, but it became exposed when vesicle formation was compromised in the sec7 mutant, and it was also exposed on secretory vesicles (SVs), as reported previously. However, flippase mutations did not reduce the exposure of PS in either case, even at low levels that would only be detectable by quantitative analysis of mRFP-Lact-C2 fluorescence in isolated SVs. Furthermore, no reduction in the PS level was observed in a mutant with multiple flippase mutations. Because PS was not exposed in a mutant that accumulates ER or cis/medial-Golgi membranes, Golgi maturation seems to be a prerequisite for PS translocation. Our results suggest that an unknown mechanism, possibly a protein with flippase-like activity, acts in conjunction with known flippases to regulate PS translocation.

Role of phosphatidylserine in phospholipid flippase-mediated vesicle transport in Saccharomyces cerevisiae.

Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.

Interaction of the phospholipid flippase Drs2p with the F-box protein Rcy1p plays an important role in early endosome to trans-Golgi network vesicle transport in yeast.

Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.

Identification of a second amphipathic lipid-packing sensor-like motif that contributes to Gcs1p function in the early endosome-to-TGN pathway.

ArfGAPs, GTPase-activating proteins for Arf small GTPases, are involved in multiple steps of vesicle formation of various transport pathways. Amphipathic lipid-packing sensor (ALPS) motif was first identified in the C-terminal regions of ArfGAP1 and its yeast homologue Gcs1p as a region that adsorbs preferentially onto highly curved membranes by folding into an amphipathic α-helix (AH). We previously showed that Gcs1p functionally interacted with the phospholipid flippase Cdc50p-Drs2p in the early endosome-to-TGN retrieval pathway. In this study, we performed functional analyses of the C-terminal region of Gcs1p containing ALPS. Hydrophobic cluster analysis suggested that there is another potential AH-forming region downstream of ALPS in Gcs1p. Mutational analysis suggested that the ALPS motif is important for the Gcs1p function in the Golgi-to-ER retrograde pathway, whereas ALPS and the predicted AH region redundantly function in the post-Golgi pathways including the early endosome-to-TGN pathway. Liposome flotation assay indicated that this downstream region preferentially interacted with liposomes of smaller size. The region containing the ALPS motif was also required for the interaction with SNARE proteins including Snc1p and Tlg1p. These results suggest that ALPS and the predicted AH region are involved in the regulation and function of Gcs1p by interacting with membrane phospholipids and vesicle proteins.

Phospholipid flippases Lem3p-Dnf1p and Lem3p-Dnf2p are involved in the sorting of the tryptophan permease Tat2p in yeast.

The type 4 P-type ATPases are flippases that generate phospholipid asymmetry in membranes. In budding yeast, heteromeric flippases, including Lem3p-Dnf1p and Lem3p-Dnf2p, translocate phospholipids to the cytoplasmic leaflet of membranes. Here, we report that Lem3p-Dnf1/2p are involved in transport of the tryptophan permease Tat2p to the plasma membrane. The lem3Δ mutant exhibited a tryptophan requirement due to the mislocalization of Tat2p to intracellular membranes. Tat2p was relocalized to the plasma membrane when trans-Golgi network (TGN)-to-endosome transport was inhibited. Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3Δ mutant. The NH(2)-terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids, including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results; Tat2p is not ubiquitinated when the NH(2)-terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3Δ mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole.

Mutations in N-terminal flanking region of blue light-sensing light-oxygen and voltage 2 (LOV2) domain disrupt its repressive activity on kinase domain in the Chlamydomonas phototropin.

Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin.