Plasma Membrane Proton ATPase Research Articles

Allele-specific expression of AP2-like ABA repressor 1 regulates iron uptake by modulating rhizosphere pH in apple.

Genetic variation within a species can result in allelic expression for natural selection or breeding efforts. Here, we identified an iron (Fe) deficiency-inducible gene, AP2-like ABA repressor 1 (MdABR1), in apple (Malus domestica). MdABR1 exhibited differential expression at the allelic level (MdABR131A and MdABR131G) in response to Fe deficiency. The W-box insertion in the promoter of MdABR131A is essential for its induced expression and its positive role under Fe deficiency stress. MdABR1 binds to the promoter of basic helix-loop-helix 105 (MdbHLH105), participating in the Fe deficiency response, and activates its transcription. MdABR131A exerts a more pronounced transcriptional activation effect on MdbHLH105. Suppression of MdABR1 expression leads to reduced rhizosphere acidification in apple, and MdABR131A exhibits allelic expression under Fe deficiency stress, which is substantially upregulated and then activates the expression of MdbHLH105, promoting the accumulation of plasma membrane proton ATPase 8 (MdAHA8) transcripts in response to proton extrusion, thereby promoting rhizosphere acidification. Therefore, variation in the ABR1 alleles results in variable gene expression and enables apple plants to exhibit a wider tolerance capability and Fe deficiency response. These findings also shed light on the molecular mechanisms of allele-specific expression in woody plants.

Exploring the role and mechanisms of the PMA gene in Aspergillus fumigatus

ABSTRACT In the realm of aspergillosis, a critical concern for immunocompromised patients facing Aspergillus fumigatus, effective management hinges on understanding fungal growth, stress resistance, and response to antifungal treatments. Our study investigates the crucial role of fungal plasma membrane proton ATPase (PMA) in nutrient absorption, intertwined with growth and antifungal susceptibility. We employed a high-throughput knockout method to create the PMA gene knockout mutant, ΔAfu-PMA1, in A. fumigatus, alongside a complementation strain. Antifungal susceptibility to triazoles was assessed by micro-dilution method and E-test, revealing decreased sensitivity to voriconazole in ΔAfu-PMA1. Comparative analysis demonstrated significant growth differences, with wild-type strain surpassing ΔAfu-PMA1 by 3.2-fold. Under oxidative stress and heightened osmotic pressure, ΔAfu-PMA1 showed notable growth defects. Loss of PMA led to increased ergosterol and decreased ATP content, alongside pH changes in the culture medium. Transcriptome sequencing unveiled revealed a reduced expression of genes associated with ribosome function, the MAPK pathway, endoplasmic reticulum, and the transport and metabolism of fats, sugars, and proteins in ΔAfu-PMA1, highlighting PMA’s regulatory role in growth and adaptation. These findings emphasise PMA as a potential target for future antifungal drugs, offering hope in combating aspergillosis.

Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation.

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K+/H+ antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K+/H+ antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane.

Genome-wide Analysis of Drought-related Genes of “Alphonso” and “Tommy Atkins” Mango (Mangifera indica L.) Cultivars

Mango (Mangifera indica L.) is one of the most popular fruit crops grown and traded globally. Mango can withstand seasonal drought periods, especially during flowering. However, prolonged drought stress can cause the weakening of the plant and may encourage injuries and diseases caused by abiotic and biotic factors. With the availability of the mango genome, genomic studies related to drought adaptation of mango can now be extensively explored. In this study, genome- wide drought-related genes (DRGs) of mango were analyzed using the whole genome sequences (WGS) of “Alphonso” and “Tommy Atkins” cultivars (PRJNA487154 and PRJNA450143, respectively). A total of 261 and 257 DRGs were identified in the genome of “Alphonso” and “Tommy Atkins,” respectively, using BLASTP. Approximately 50% of these genes are involved in both molecular and physiological adaptations of mango to drought. Among the drought stress genes, the ABC transporter gene ABCG40 had the highest number of homologs in mango, followed by calcium-dependent protein kinase genes ZmCPK4, CPK21, and CDPK7, as well as the plasma membrane proton ATPase OST2. Gene ontology (GO) analysis of the DRGs revealed that protein binding, ATP binding, and mRNA binding are the most common molecular functions, whereas the predominant biological process of these DRGs is related to their response to water deprivation. Phylogenetic analysis showed that the drought-related proteins in “Alphonso” and “Tommy Atkins” are broadly clustered into seven and six major clades, respectively. This study, to date, offers the most comprehensive information on the genome-wide DRGs of the mango, which can strengthen marker-assisted breeding programs for drought tolerance in mango and other related fruit trees, as well as the future incorporation of favorable alleles toward improving the overall agronomic characteristics of this Philippine important fruit crop.

Promotion and Upregulation of a Plasma Membrane Proton-ATPase Strategy: Principles and Applications.

Plasma membrane proton-ATPase (PM H+-ATPase) is a primary H+ transporter that consumes ATP in vivo and is a limiting factor in the blue light-induced stomatal opening signaling pathway. It was recently reported that manipulation of PM H+-ATPase in stomatal guard cells and other tissues greatly improved leaf photosynthesis and plant growth. In this report, we review and discuss the function of PM H+-ATPase in the context of the promotion and upregulation H+-ATPase strategy, including associated principles pertaining to enhanced stomatal opening, environmental plasticity, and potential applications in crops and nanotechnology. We highlight the great potential of the promotion and upregulation H+-ATPase strategy, and explain why it may be applied in many crops in the future.

A long non-coding apple RNA, MSTRG.85814.11, acts as a transcriptional enhancer of SAUR32 and contributes to the Fe-deficiency response.

Iron (Fe) is an essential plant nutrient and its deficiency typically limits plant growth. Long non-coding (lnc) RNAs are involved in adaptive responses to nutrient stress; however, it is not known whether they function in the regulation of the canonical Fe-deficiency response. The expression of Malus domestica (apple) lncRNA MSTRG.85814 is induced by Fe deficiency, as identified by high-throughput strand-specific RNA-seq analysis of an apple homograft system. MSTRG.85814 has a complex structure, with 13 predicted RNA sequence variants, four of which are upregulated in the roots of plants experiencing Fe deficiency. We found that one MSTRG.85814 splice variant (MSTRG.85814.11) positively modulated its cis target mRNA derived from the small auxin upregulated gene SAUR32. This in turn promoted the expression of SAUR32 and caused an increase in the expression of a plasma membrane proton ATPase, AHA10. Using a pH imaging technique, a significant decrease in the apoplastic pH was observed to occur in the root tips of MSTRG.85814.11 or SAUR32-overexpressing apple plants. Thus MSTRG.85814.11 was shown to positively promote SAUR32 expression, which then activated proton extrusion involved in the Fe-deficiency response. These results reveal a mechanism by which lncRNA promotes environmental Fe-deficiency stress adaption.

Transcriptome profiling of wheat genotypes under heat stress during grain-filling

High temperatures adversely impact productivity and quality (nutritional and functional) of wheat grains. Anthesis and post-anthesis developmental stages are more sensitive to heat stress, with the grain-filling stage being crucial for sustained grain yield and quality. Comparative transcriptome profiling of the developing grain of three wheat genotypes (contrasting for heat stress tolerance) during early- (14-dpa (days post anthesis)) and late-grain filling (30-dpa) was studied to identify key genes involved in imparting heat tolerance. Heat stress during grain-filling (early- and late-) significantly down-regulated key genes in the genotypes, Gregory and Banks, found to be more heat susceptible. Upregulation of the cluster of genes comprising 6-phosphogluconate dehydrogenase, S6 RPS6-2 ribosomal protein, peptidylprolyl isomerase, plasma membrane proton ATPase, Heat shock cognate-70, FtsH protease, RuBisCO activase B, methionine synthase, cytochrome C (class I), and HMW-glutenin in the genotype Fang-60 during heat stress was found to be associated with heat stress tolerance in this genotype. Upregulation of β-glucanase (1,3 & 1,4), triose phosphate isomerase and calnexin at 14-dpa; and downregulation of Na+/H+antiporter, glucose-1-phosphate adenylyltransferase, ips1 riboregulator and AraC family transcriptional regulator at 30-dpa was observed specifically in the heat susceptible genotypes. Hsp-family, ascorbate peroxidase, β-amylase, γ-gliadin-2 and LMW-glutenin were heat stress responsive and were upregulated during stress across 14- and 30-dpa in all three genotypes. This study provides insights into genes that may be involved in regulating heat tolerance in a tolerant genotype and those that are responsive to heat in the developing wheat grain of tolerant and susceptible genotypes. The genotype Fang-60 was demonstrated to be a potential source of heat stress tolerance for use in wheat breeding.

Novel Zinc-Attenuating Compounds as Potent Broad-Spectrum Antifungal Agents with In Vitro and In Vivo Efficacy.

ABSTRACTAn increase in the incidence of rare but hard-to-treat invasive fungal pathogens as well as resistance to the currently available antifungal drugs calls for new broad-spectrum antifungals with a novel mechanism of action. Here we report the identification and characterization of two novel zinc-attenuating compounds, ZAC307 and ZAC989, which exhibit broad-spectrum in vitro antifungal activity and in vivo efficacy in a fungal kidney burden candidiasis model. The compounds were identified serendipitously as part of a drug discovery process aimed at finding novel inhibitors of the fungal plasma membrane proton ATPase Pma1. Based on their structure, we hypothesized that they might act as zinc chelators. Indeed, both fluorescence-based affinity determination and potentiometric assays revealed these compounds, subsequently termed zinc-attenuating compounds (ZACs), to have strong affinity for zinc, and their growth inhibitory effects on Candida albicans and Aspergillus fumigatus could be inactivated by the addition of exogenous zinc to fungal growth media. We determined the ZACs to be fungistatic, with a low propensity for resistance development. Gene expression analysis suggested that the ZACs interfere negatively with the expression of genes encoding the major components of the A. fumigatus zinc uptake system, thus supporting perturbance of zinc homeostasis as the likely mode of action. With demonstrated in vitro and in vivo antifungal activity, low propensity for resistance development, and a novel mode of action, the ZACs represent a promising new class of antifungal compounds, and their advancement in a drug development program is therefore warranted.

Two novel effectors of trafficking and maturation of the yeast plasma membrane H+ -ATPase.

The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system. Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.

Abstract 5429: The Warburg Effect: protons suck

Abstract Aerobic glycolysis (the Warburg Effect) is a hallmark of cancer and is associated with local invasion and metastasis. This metabolic phenotype results in acidification of the microenvironment in solid tumors, which is responsible for many of the known sequelae. For example, systemic buffer therapy directly and specifically increases extracellular tumor pH and reduces spontaneous and experimental metastasis in vivo. Further, extracellular acidosis can be a potent inhibitor of anti-tumor immunity. Removal of glycolytically-derived acids requires the activity of proton transporting mechanisms, such as NHE, V-ATPase and CA-IX. While it is commonly believed that these transporters are responding to the demands imposed by increased glycolytic flux, an alternative hypothesis states that glycolytic flux is increased to satisfy the demand driven by these transporters. Hence, expression of transporters that drive extracellular acidosis would induce a more glycolytic phenotype. To test this, we have transfected lowly glycolytic and non-metastatic MCF-7 breast adenocarcinoma cells with two different proton transporters. In the first model, we used the yeast plasma membrane proton ATPase 1 (PMA1); a Type 1 P-type ATPase. PMA1 was stably over-expressed in MCF7 cell line and over-expression of PMA1 transformed the cells into a more aggressive phenotype with increased glucose consumption, lactate production, and proton production. Phenotypically, the transfected clones had elevated rates of migration and invasion in vitro and increased metastasis in vivo. This experimental evidence supports the hypothesis that proton export alone could drive the Warburg phenotype. Hence, in this system, glucose is metabolized to replenish either the ATP expended or the exported hydrogen-ions. In the second model we used CA-IX; an exofacial carbonic anhydrase that is highly expressed in invasive cancers and materially participates in acidifying the microenvironment. CA-IX is upregulated in multiple cancer types with minimal expression in normal tissue, and is therefore an attractive and biologically relevant therapeutic target. In vitro, we have observed that many cells expressing a glycolytic phenotype have elevated CA-IX expression, leading us to postulate that these phenotypes are coupled. To test this, we stably overexpressed CA-IX in MCF7 cells and again this resulted in a stable, glycolytic phenotype with increased glucose consumption, lactate production, and proton production observed using assay kits and live cell metabolic phenotyping with the Seahorse Extracellular Flux Analyzer. Through this study we have shown that acidosis leads to a more aggressive behavior and that CA-IX expression alone can induce a glycolytic phenotype. Hence, we hypothesise this is due to the enzymatic activity of CA-IX “sucking in” glucose to replenish intracellular H+ that are exported by this mechanism. Citation Format: Shonagh Russell, Liping Xu, Rober J. Gillies. The Warburg Effect: protons suck [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5429. doi:10.1158/1538-7445.AM2017-5429

Mother-daughter asymmetry of pH underlies aging and rejuvenation in yeast.

Replicative aging in yeast is asymmetric-mother cells age but their daughter cells are rejuvenated. Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation. Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells. This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1). Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells. We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify. We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons. However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation.

Fumonisinas –Síntesis y función en la interacción Fusarium verticillioides-maíz

Fusarium verticillioides is the main fungal pathogen that affects the productivity of maize worldwide. The fungus penetrates the plant by different routes and infects roots, stem and cob. The pathogen produces several toxins in tissue and corn kernels, which affect their quality. Fumonisins are the major toxins produced by this fungus. The ability to produce them depends on the presence of several genes encoding the enzymes responsible for biosynthesis. The regulation of the synthesis is very complex and depends on environmental and nutritional factors, as well as multiple signaling pathways. This is reflected by the high variability in fumonisin production among F. verticillioides strains. Fumonisins are virulence factors because their production is associated with a greater capacity to infect maize seedlings. However, this role is not clear for ear infection and rotting. In maize, fumonisins have three molecular targets: sphinganine N-acyl-transferase, plasma membrane proton ATPase and the basic β-1,3-glucanases. These three enzymes have important physiological functions and also participate in the plant defense response against fungal pathogens.

Expression of a Translationally Fused TAP-Tagged Plasma Membrane Proton Pump in Arabidopsis thaliana

The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5′ end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry.

Plasma membrane H+‐ATPase gene expression, protein level and activity in growing and non‐growing regions of barley (Hordeum vulgare) leaves

Plasma membrane proton ATPase (PM-H⁺-ATPase) is the key means through which plant cells energize nutrient uptake and acidify the apoplast. Both of these processes aid cell elongation; yet, it is not known how such a suspected role of the PM-H⁺-ATPase in growth is reflected through changes in its transcript level and activity in grass leaves. In the present study on leaf three of barley, the elongation zone and the emerged blade, which contained fully expanded cells were analyzed. Plasma membranes were isolated and used to assay the activity (ATPase assay) and abundance (western blotting) of PM-H⁺-ATPase protein. Expression of mRNA was quantified using real-time polymerase chain reaction (qPCR). PM-H⁺-ATPase transcript and protein level and activity differed little between growing and non-growing leaf regions when values were related to unit extracted total RNA and cell number, respectively. However, when values were related to unit surface area of plasma membrane, they were more than twice as high in growing compared with non-growing leaf tissue. It is concluded that this higher surface density of PM-H⁺-ATPase activity in growing barley leaf tissue aids apoplast acidification and cell expansion.

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mβCD). Removal of ergosterol by mβCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mβCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mβCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.

Osmoregulation of leaf motor cells

“Osmotic Motors” – the best-documented explanation for plant leaf movements – frequently reside in specialized motor leaf organs, pulvini. The movements result from dissimilar volume and turgor changes in two oppositely positioned parts of the pulvinus. This Osmotic Motor is powered by a plasma membrane proton ATPase, which drives KCl fluxes and, consequently, water, across the pulvinus into swelling cells and out of shrinking cells. Light signals and signals from the endogenous biological clock converge on the channels through which these fluxes occur. These channels and their regulatory pathways in the pulvinus are the topic of this review.

Micromolar HgCl 2 concentrations transitorily duplicate the ATP level in Saccharomyces cerevisiae cells

Low concentrations of HgCl 2 elicited, in Saccharomyces cerevisiae, a transitory increase in the ATP level followed by a decrease of its concentration, until almost disappearance. At 1 μM HgCl 2, the increase in ATP lasted for about 30 min, while at 10 μM the increase was only observed in the first 5 min of treatment. The initial burst of ATP was accompanied by a decrease in the level of hexose phosphates, whereas during the decrease of ATP an increase in the inosine and hexose phosphates levels took place. The treatment with HgCl 2 inhibited the plasma membrane proton ATPase but not the activities of hexokinase or 6-phosphofructokinase.

Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast.

Correct sorting of proteins is essential to generate and maintain the identity and function of the different cellular compartments. In this study we demonstrate the role of lipid rafts in biosynthetic delivery of Pma1p, the major plasma membrane proton ATPase, to the cell surface. Disruption of rafts led to mistargeting of Pma1p to the vacuole. Conversely, Pma1-7, an ATPase mutant that is mistargeted to the vacuole, was shown to exhibit impaired raft association. One of the previously identified suppressors, multicopy AST1, not only restored surface delivery but also raft association of Pma1-7. Ast1p, which is a peripheral membrane protein, was found to directly interact with Pma1p inducing its clustering into a SDS/Triton X100-resistant oligomer. We suggest that clustering facilitates partition of Pma1p into rafts and transport to the cell surface.

Identification by RNA fingerprinting of genes differentially expressed during the development of the basidiomycete Lentinula edodes.

As part of an ongoing project to understand the molecular mechanisms of fruit body development in Lentinula edodes (Shiitake mushroom), RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) was used to identify differentially expressed genes in RNA populations from four stages of L. edodes development vegetative mycelium, primordium, young fruit body and mature fruit body. From 30 RNA fingerprints, we cloned and sequenced 33 RAP fragments after their differential expression patterns had been verified by reverse Northern dot-blot hybridization. Thirteen RAP fragments show high sequence similarity to known gene products which are involved in (1) transport across the plasma membrane (drug efflux pump and sugar transporter); (2) cell cycle control (cyclin B); (3) signal transduction and transcriptional regulation (mitogen-activated protein kinase, Cdc39/Not1, PriA, Jun-D); (4) intracellular molecule trafficking (ubiquitin, plasma membrane proton ATPase, and alpha-adaptin); (5) mitochondrial biogenesis (mitochondrial processing peptidase beta-subunit, mitochondrial glycerol-3-phosphate dehydrogenase); and (6) intermediary metabolism (fructose 1,6 bisphosphatase). The transcript levels for plasma membrane proton ATPase and alpha-adaptin remained constant, whereas the other eleven genes were differentially expressed during L. edodes. development. The expression profiles of the genes suggest that transport across the plasma membrane is important in the mycelial stage. Specific signal transduction and transcriptional controls may play important roles during the initiation of primordia and the formation of young fruiting bodies. When the mushroom matures, expression of genes involved in metabolic pathways becomes prominent. The isolation of these genes indicates their involvement in homobasidiomycete development and suggests new directions for molecular studies on mechanisms of mushroom development.

LST1 is a SEC24 homologue used for selective export of the plasma membrane ATPase from the endoplasmic reticulum.

In Saccharomyces cerevisiae, vesicles that carry proteins from the ER to the Golgi compartment are encapsulated by COPII coat proteins. We identified mutations in ten genes, designated LST (lethal with sec-thirteen), that were lethal in combination with the COPII mutation sec13-1. LST1 showed synthetic-lethal interactions with the complete set of COPII genes, indicating that LST1 encodes a new COPII function. LST1 codes for a protein similar in sequence to the COPII subunit Sec24p. Like Sec24p, Lst1p is a peripheral ER membrane protein that binds to the COPII subunit Sec23p. Chromosomal deletion of LST1 is not lethal, but inhibits transport of the plasma membrane proton-ATPase (Pma1p) to the cell surface, causing poor growth on media of low pH. Localization by both immunofluorescence microscopy and cell fractionation shows that the export of Pma1p from the ER is impaired in lst1Delta mutants. Transport of other proteins from the ER was not affected by lst1Delta, nor was Pma1p transport found to be particularly sensitive to other COPII defects. Together, these findings suggest that a specialized form of the COPII coat subunit, with Lst1p in place of Sec24p, is used for the efficient packaging of Pma1p into vesicles derived from the ER.