Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc)

Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, we establish that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. We show here that Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones, which is required for topographic map formation.     

Cloning and Tissue Expression of 6-Pyruvoyl Tetrahydropterin Synthase and Xanthine Dehydrogenase from <Emphasis Type="Italic">Poecilia reticulata</Emphasis>

Guppy is a popular ornamental fish owing to its diverse body and fin coloration. More than 40 established color varieties have been selectively bred. The complementary DNAs for 2 enzymes that are involved in the de novo synthesis of pteridines and purines, which are important for the production of color pigments, were cloned from the caudal fin. Two cDNA isoforms for 6-pyruvoyl tetrahydropterin synthase (PTPS), with an open reading frame of 130 and 147 amino acids, respectively, were cloned from the Red Tail variety. The deduced amino acid sequence of the longer isoform shows an overall identity of about 65% to the mammalian PTPS sequences. The cDNA for xanthine dehydrogenase (XDH) was cloned from the Yellow Tail variety, and consists of an open reading frame of 1331 amino acids. Although it shows a higher overall identity to bovine aldehyde oxidase (AO; 54%) than to chicken XDH (51%), it has a NAD-binding domain that is specific to XDHs. Northern blot analysis indicated that both PTPS and XDH messenger RNAs were highly expressed in the liver, but absent in the muscle. In the caudal fins, guppy varieties with a higher proportion of xanthophores and erythrophores showed higher expression of PTPS, while XDH mRNA levels were too low to indicate obvious differential expression among the color guppy varieties. The results implied that high expression of PTPS is correlated with the biosynthesis of pteridines in the erythrophores and xanthophores, while the association between the putative guppy XDH with specific chromatophores is less clear.     

D-threo-tetrahydrobiopterin is synthesized via 1'-oxo-2'-D-hydroxypropyl-tetrahydropterin in Dictyostelium discoideum Ax2

The biosynthesis of D-threo-tetrahydrobiopterin (DH4, tetrahydrodictyopterin) in Dictyostelium discoideum Ax2 was investigated through the mutant disrupted in the gene encoding sepiapterin reductase (SR) by insertional inactivation. The mutant cells, being completely devoid of SR protein, showed 18.1% of L-erythro-tetrahydrobiopterin (BH4) and 0.6% of DH4 productions in the wild type cells. The mutant cells were also identified to excrete D- and L-sepiapterin, which were presumed to originate from intracellular 1'-oxo-2'-D-hydroxypropyl- and 1'-oxo-2'-L-hydroxypropyl-tetrahydropterin (H4-pterin), respectively. Furthermore, in a coupled assay with Dictyostelium SR, the mutant cell extract exhibited a novel enzyme activity converting 6-pyruvoyltetrahydropterin to 1'-oxo-2'-D-hydroxypropyl-H4-pterin. These results are clear demonstration of the in vivo synthesis of DH4 via 1'-oxo-2'-D-hydroxypropyl-H4-pterin as well as an alternative synthesis of BH4 and DH4 in the complete absence of SR.     

A mutation in the silver gene leads to defects in melanosome biogenesis and alterations in the visual system in the zebrafish mutant fading vision

Forward genetic screens have been instrumental in defining molecular components of visual function. The zebrafish mutant fading vision (fdv) has been identified in such a screen due to defects in vision accompanied by hypopigmentation in the retinal pigment epithelium (RPE) and body melanocytes. The RPE forms the outer most layer of the retina, and its function is essential for vision. In fdv mutant larvae, the outer segments of photoreceptors are strongly reduced in length or absent due to defects in RPE cells. Ultrastructural analysis of RPE cells reveals dramatic cellular changes such as an absence of microvilli and vesicular inclusions. The retinoid profile is altered as judged by biochemical analysis, arguing for a partial block in visual pigment regeneration. Surprisingly, homozygous fdv vision mutants survive to adulthood and show, despite a persistence of the hypopigmentation, a partial recovery of retinal morphology. By positional cloning and subsequent morpholino knock-down, we identified a mutation in the silver gene as the molecular defect underlying the fdv phenotype. The Silver protein is required for intralumenal fibril formation in melanosomes by amylogenic cleavage. Our data reveal an unexpected link between melanosome biogenesis and the visual system, undetectable in cell culture.     

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q₆ increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q₆ or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q₆, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.     

Synaptic polarity depends on phosphatidylinositol signaling regulated by myo-inositol monophosphatase in Caenorhabditis elegans

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP(2) on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP(2) synthesis through inhibition of IMPase. We propose that the PIP(2) signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity.     

Rice <Emphasis Type="Italic">phot1a</Emphasis> mutation reduces plant growth by affecting photosynthetic responses to light during early seedling growth

The aim of this work was to characterize the phot1 mutant of rice during early seedling growth in various light conditions. We isolated the rice T-DNA insertion mutant phot1a-1 and compared it to the Tos17 insertion mutant phot1a-2. When phot1a mutants were grown under WL (100) and BL (40 μmol m⁻² s⁻¹), they demonstrated a considerable reduction in photosynthetic capacity, which included decreased leaf CO₂ uptake and plant growth. Pigment analysis showed no significant difference between wild-type and mutants in the Chl a:b ratios, whereas in the latter, total concentration was reduced (a 2-fold decrease). Carotenoid contents of the mutants were also decreased considerably, implying the involvement of phot1a in pigment degradation. Deletion of phot1a showed higher contents of H₂O₂ in leaves. Chloroplastic APX and SOD activities were lower in the mutants whereas the activities of cytosolic enzymes were increased. Immunoblotting indicated reduced accumulation of photosystem proteins (D1, D2, CP43, Lhca2, and PsaC) relative to the other light-harvesting complexes in the mutant. We conclude that the defect of Os Phot1a affects degradation of chlorophylls and carotenoids, and under photosynthetically active photon fluxes, mutation of phot1a results in loss of photosynthetic capacity owing to the damage of photosystems caused by elevated H₂O₂ accumulation, leading to a reduction in plant growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Release of the export adapter, Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplasmic GTPase Lsg1p

In eukaryotes, nuclear export of the large (60S) ribosomal subunit requires the adapter protein Nmd3p to provide the nuclear export signal. Here, we show that in yeast release of Nmd3p from 60S subunits in the cytoplasm requires the ribosomal protein Rpl10p and the G-protein, Lsg1p. Mutations in LSG1 or RPL10 blocked Nmd3-GFP shuttling into the nucleus and export of pre-60S subunits from the nucleus. Overexpression of NMD3 alleviated the export defect, indicating that the block in 60S export in lsg1 and rpl10 mutants results indirectly from failing to recycle Nmd3p. The defect in Nmd3p recycling and the block in 60S export in both lsg1 and rpl10 mutants was also suppressed by mutant Nmd3 proteins that showed reduced binding to 60S subunits in vitro. We propose that the correct loading of Rpl10p into 60S subunits is required for the release of Nmd3p from subunits by Lsg1p. These results suggest a coupling between recycling the 60S export adapter and activation of 60S subunits for translation.     

Cuticle Integrity and Biogenic Amine Synthesis in Caenorhabditis elegans Require the Cofactor Tetrahydrobiopterin (BH4)

Tetrahydrobiopterin (BH4) is the natural cofactor of several enzymes widely distributed among eukaryotes, including aromatic amino acid hydroxylases (AAAHs), nitric oxide synthases (NOSs), and alkylglycerol monooxygenase (AGMO). We show here that the nematode Caenorhabditis elegans, which has three AAAH genes and one AGMO gene, contains BH4 and has genes that function in BH4 synthesis and regeneration. Knockout mutants for putative BH4 synthetic enzyme genes lack the predicted enzymatic activities, synthesize no BH4, and have indistinguishable behavioral and neurotransmitter phenotypes, including serotonin and dopamine deficiency. The BH4 regeneration enzymes are not required for steady-state levels of biogenic amines, but become rate limiting in conditions of reduced BH4 synthesis. BH4-deficient mutants also have a fragile cuticle and are generally hypersensitive to exogenous agents, a phenotype that is not due to AAAH deficiency, but rather to dysfunction in the lipid metabolic enzyme AGMO, which is expressed in the epidermis. Loss of AGMO or BH4 synthesis also specifically alters the sensitivity of C. elegans to bacterial pathogens, revealing a cuticular function for AGMO-dependent lipid metabolism in host–pathogen interactions.     

Molecular determinants for FMN-binding in Desulfovibrio gigas flavoredoxin

Flavoredoxin participates in Desulfovibrio gigas thiosulfate reduction pathway. Its 3-dimensional model was generated allowing the oxidized riboflavin-5'-phosphate (FMN) site to be predicted. Residues likely to be involved in FMN-binding were identified (N29, W35, T56, K92, H131 and F164) and mutated to alanine. Fluorescence titration with apoprotein showed that FMN is strongly bound in the wild-type protein. Comparison of K(d) values for mutants suggests that interactions with the phosphate group of FMN, contribute more to binding than the interactions with the isoalloxazine ring. The redox potential of bound FMN determined for wild-type and mutants revealed shifts to less negative values. These findings were correlated with the protein structure in order to contribute to a better understanding of the structure-function relationships in flavoredoxin.     

A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae

We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.     

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.     

HPLC analysis of tetrahydrobiopterin and its pteridine derivatives using sequential electrochemical and fluorimetric detection: application to tetrahydrobiopterin autoxidation and chemical oxidation

Tetrahydrobiopterin (BH(4)) is an essential cofactor of endothelial nitric oxide (NO) synthase and when depleted, endothelial dysfunction results with decreased production of NO. BH(4) is also an anti-oxidant being a good "scavenger" of oxidative species. NADPH oxidase, xanthine oxidase, and mitochondrial enzymes producing reactive oxygen species (ROS) can induce elevated oxidant stress and cause BH(4) oxidation and subsequent decrease in NO production and bioavailability. In order to define the process of ROS-mediated BH(4) degradation, a sensitive method for monitoring pteridine redox-state changes is required. Considering that the conventional fluorescence method is an indirect method requiring conversion of all pteridines to oxidized forms, it would be beneficial to use a rapid quantitative assay for the individual detection of BH(4) and its related pteridine metabolites. To study, in detail, the BH(4) oxidative pathways, a rapid direct sensitive HPLC assay of BH(4) and its pteridine derivatives was adapted using sequential electrochemical and fluorimetric detection. We examined BH(4) autoxidation, hydrogen peroxide- and superoxide-driven oxidation, and Fenton reaction hydroxyl radical-driven BH(4) transformation. We demonstrate that the formation of the primary two-electron oxidation product, dihydrobiopterin (BH(2)), predominates with oxygen-induced BH(4) autoxidation and superoxide-catalyzed oxidation, while the irreversible metabolites, pterin and dihydroxanthopterin (XH(2)), are largely produced during hydroxyl radical-driven BH(4) oxidation.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

The pteridine pathway in zebrafish: regulation and specification during the determination of neural crest cell-fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72-h postfertilization, producing H4biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6-lactoyl-7,8-dihydropterin) accumulates as yellow pigment in xanthophores, together with 7-oxobiopterin, isoxanthopterin and 2,4,7-trioxopteridine. Sepiapterin is the key intermediate in the formation of 7-oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42-h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP-H4biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest-derived cells and the regulation of pteridine synthesis in mammals.     

Regulation of tyrosinase by tetrahydropteridines and H2O2

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.     

Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH

myo-Inositol is important for cell signaling both in cytoplasm and in intracellular organelles. It is required in the plasma membrane and cytoplasm for maintained synthesis of the second messengers, inositoltrisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP(2)), and in organelles as precursor for synthesis of complex signaling phospholipids and inositolphosphates from IP(3) and PIP(2). myo-Inositol must be taken up into the cell where its is used, because neither neurons nor astrocytes synthesize it. It is also an osmolyte, taken up in response to surrounding hyperosmolarity and released during hypo-osmolarity. There are three myo-inositol transporters, the Na(+)-dependent SMIT1 and SMIT2, and HMIT, which co-transports myo-inositol with H(+). Their relative expressions in astrocytes and neurons are unknown. Uptake kinetics for myo-inositol in astrocytes has repeatedly been determined, but always on the assumption of only one component, leaving kinetics for the individual transporters unknown. This paper demonstrates that astrocytes obtained directly from the brain express SMIT1 and HMIT, but little SMIT2, and that all three transporters are expressed in neurons. Cultured mouse astrocytes show a high-affinity/low-capacity myo-inositol uptake (V(max): 60.0 ± 3.0 pmol/min per mg protein; K(m): 16.7 ± 2.6 μM), mediated by SMIT1 and perhaps partly by SMIT2. It was determined in cells pre-treated with HMIT-siRNA and confirmed by specific inhibition of SMIT. However at physiologically relevant myo-inositol concentrations most uptake is by a lower-affinity/higher-capacity uptake, mediated by HMIT (V(max): 358 ± 60 pmol/min per mg protein; K(m): 143 ± 36 μM) and determined by subtraction of SMIT-mediated from total uptake. At high myo-inositol concentrations, its uptake is inhibited by incubation in medium with increased pH, and increased during intracellular acidification with NH(4)Cl. This is in agreement with literature data for HMIT alone. At low concentration, where SMIT1/2 activity gains importance, myo-inositol uptake is reduced by ammonia-induced intracellular acidification, consistent with the transporter's pH sensitivity reported in the literature.     

Fab1p Is Essential for PtdIns(3)P 5-Kinase Activity and the Maintenance of Vacuolar Size and Membrane Homeostasis

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH₂-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P₂. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1^tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P₂, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P₂ synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P₂ synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P₂. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P₂ by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P₂ has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P₂, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P₂.