Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis . A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M₂ generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2 , are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT:: Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represent a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis , and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.     

Epigenetic instability and trans-silencing interactions associated with an SPT::Ac T-DNA locus in tobacco.

Progeny of tobacco line 2853.6, which carries a streptomycin phosphotransferase (SPT) gene interrupted by the maize element Activator (Ac), were selected for streptomycin resistance (Spr) because of germinal Ac excision. Some events gave rise to Spr alleles that were unstable and exhibited a mottled phenotype on streptomycin-containing medium due to somatic loss of SPT function. This instability was most pronounced in one particular line, Spr12F. Other Spr alleles rarely exhibited silencing of SPT. Streptomycin-sensitive, homozygous Spr12F plants were recovered, and crosses were performed with other, more stable Spr lines. A high proportion of the resulting heterozygous progeny were silenced for SPT expression. The silenced state was heritable even after the Spr12F allele segregated away. No correlation could be made between silencing and methylation of the SPTgene. Structural analysis of allele Spr12F showed that the SPT gene from which Ac had excised was flanked by direct repeats of Ac. A search was carried out among 110 additional Spr alleles for new independent unstable alleles, and four were identified. All of these alleles also carried an SPT gene flanked by direct repeats of Ac. Thus, there is a strong correlation between this structure and instability of SPT expression.     

Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1-3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.     

Cloning and initial characterization of a new subunit for mammalian serine-palmitoyltransferase

Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2- to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis.     

The Ca(2+)/Calcineurin-Dependent Signaling Pathway in the Gray Mold Botrytis cinerea: The Role of Calcipressin in Modulating Calcineurin Activity

In the gray mold fungus Botrytis cinerea the Gα subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/calmodulin-dependent phosphatase calcineurin. In this study we focused on the functional characterization of the catalytic subunit of calcineurin (BcCnA) and its putative regulator calcipressin (BcRcn1). We deleted the genes encoding both proteins to examine their role concerning growth, differentiation and virulence. The ΔbccnA mutant shows a severe growth defect, does not produce conidia and is avirulent, while the loss of BcRcn1 caused retardation of hyphal growth and delayed infection of host plants, but had no impact on conidiation and sclerotia formation. Expression of several calcineurin-dependent genes and bccnA itself is positively affected by BcRcn1. Complementation of the Δbcrcn1 mutant with a GFP-BcRcn1 fusion construct revealed that BcRcn1 is localized in the cytoplasm and accumulates around the nuclei. Furthermore, we showed that BcCnA physically interacts with BcRcn1 and the regulatory subunit of calcineurin, BcCnB. We investigated the impact of several protein domains characteristic for modulation and activation of BcCnA via BcRcn1, such as the phosphorylation sites and the calcineurin-docking site, by physical interaction studies between BcCnA and wild-type and mutated copies of BcRcn1. Based on the observed phenotypes we conclude that BcRcn1 acts as a positive modulator of BcCnA and the Ca(2+)/calcineurin-mediated signal transduction in B. cinerea, and that both proteins regulate fungal development and virulence.     

Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation

A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.     

The Botrytis cinerea phytotoxin botcinic acid requires two polyketide synthases for production and has a redundant role in virulence with botrydial

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.     

Variable Patterns of Transposition of the Maize Element Activator in Tobacco

The strategy to be followed in a transposon tagging experiment will be determined largely by the transposition pattern of the transposon in question. With a view to utilizing the maize element Activator (Ac) as a transposon tag in heterologous systems, we investigated the pattern of Ac transposition from six different loci in transgenic tobacco. We isolated germinal revertants from plants carrying mutable alleles of the antibiotic-resistant gene streptomycin phosphotransferase (SPT) and mapped the location of the transposed Ac (trAc) elements relative to the donor SPT gene. A comparison of the distributions of trAcs among the six loci revealed that, although the receptor sites for trAcs tend to be linked to the donor locus, the pattern of Ac transposition in tobacco displays surprising locus-to-locus variation. Some trAc distributions showed the same tight clustering around the donor locus previously seen in maize, whereas others were more dispersed. The possible meaning of these findings and their implication for transposon tagging in heterologous systems are discussed.     

Endotoxin Conditioning Induces VCP/p97-mediated and Inducible Nitric-oxide Synthase-dependent Tyr284 Nitration in Protein Phosphatase 2A

Endotoxins activate Toll-like receptors and reprogram cells to be refractory to secondary exposure. Here we found that activation of different Toll-like receptors elicited a time- and dose-dependent increase in the levels of the protein phosphatase 2A catalytic subunit (PP2Ac) but not its partner A subunit. We purified the lipopolysaccharide-induced form of PP2A by chromatography plus immunoprecipitation and used mass spectrometry to identify VCP/p97 as a novel partner for PP2Ac. Endogenous VCP/p97 and PP2Ac were co-immunoprecipitated from primary murine macrophages and human lymphocytes. GST-VCP/p97 bound purified PP2A in pulldown assays, showing direct protein-protein interaction. Endotoxin conditioning of macrophages induced formation of 3-nitrotyrosine in the PP2Ac associated with VCP/p97, a response severely reduced in macrophages from iNOS knock-out mice. The reaction of purified PP2A with peroxynitrite dissociated the A subunit, and 3-nitro-Tyr²⁸⁴ was identified in PP2Ac by mass spectrometry. Myc-PP2Ac (Y284F) expressed in cells was resistant to peroxynitrite-induced nitration and reduction of A subunit binding. Transient expression of either VCP/p97 or PP2Ac was sufficient to elevate levels of the dual specificity phosphatase DUSP1, reduce p38 MAPK activation, and suppress tumor necrosis factor-α release. We propose that VCP/p97-mediated Tyr nitration of PP2A increases the levels of phosphatases PP2A and DUSP1 to contribute to the refractory response of conditioned cells.     

V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

The vacuolar (H⁺)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing.     

The structure of Tap42/alpha4 reveals a tetratricopeptide repeat-like fold and provides insights into PP2A regulation

Physiological functions of protein phosphatase 2A (PP2A) are determined via the association of its catalytic subunit (PP2Ac) with diverse regulatory subunits. The predominant form of PP2Ac assembles into a heterotrimer comprising the scaffolding PR65/A subunit together with a variable regulatory B subunit. A distinct population of PP2Ac associates with the Tap42/alpha4 subunit, an interaction mutually exclusive with that of PR65/A. Tap42/alpha4 is also an interacting subunit of the PP2Ac-related phosphatases, PP4 and PP6. Tap42/alpha4, an essential protein in yeast and suppressor of apoptosis in mammals, contributes to critical cellular functions including the Tor signaling pathway. Here, we describe the crystal structure of the PP2Ac-interaction domain of Saccharomyces cerevisiae Tap42. The structure reveals an all alpha-helical protein with striking similarity to 14-3-3 and tetratricopeptide repeat (TPR) proteins. Mutational analyses of structurally conserved regions of Tap42/alpha4 identified a positively charged region critical for its interactions with PP2Ac. We propose a scaffolding function for Tap42/alpha4 whereby the interaction of PP2Ac at its N-terminus promotes the dephosphorylation of substrates recruited to the C-terminal region of the molecule.     

Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.     

Overlapping binding sites in protein phosphatase 2A for association with regulatory A and alpha-4 (mTap42) subunits

Diverse functions of protein Ser/Thr phosphatases depend on the distribution of the catalytic subunits among multiple regulatory subunits. In cells protein phosphatase 2A catalytic subunit (PP2Ac) mostly binds to a scaffold subunit (A subunit or PR65); however, PP2Ac alternatively binds to alpha-4, a subunit related to yeast Tap42 protein, which also associates with phosphatases PP4 or PP6. We mapped alpha-4 binding to PP2Ac to the helical domain, residues 19-165. We mutated selected residues and transiently expressed epitope-tagged PP2Ac to assay for association with A and alpha-4 subunits by co-precipitation. The disabling H118N mutation at the active site or the presence of the active site inhibitor microcystin-LR did not interfere with binding of PP2Ac to either the A subunit or alpha-4, showing that these are allosteric regulators. Positively charged side chains Lys(41), Arg(49), and Lys(74) on the back surface of PP2Ac are unique to PP2Ac, compared with phosphatases PP4, PP6, and PP1. Substitution of one, two, or three of these residues with Ala produced a progressive loss of binding to the A subunit, with a corresponding increase in binding to alpha-4. Conversely, mutation of Glu(42) in PP2Ac essentially eliminated PP2Ac binding to alpha-4, with an increase in binding to the A subunit. Reciprocal changes in binding because of mutations indicate competitive distribution of PP2Ac between these regulatory subunits and demonstrate that the mutated catalytic subunits retained a native conformation. Furthermore, neither the Lys(41)-Arg(49)-Lys(74) nor Glu(42) mutations affected the phosphatase-specific activity or binding to microcystin-agarose. Binding of PP2Ac to microcystin and to alpha-4 increased with temperature, consistent with an activation energy barrier for these interactions. Our results reveal that the A subunit and alpha-4 (mTap42) require charged residues in separate but overlapping surface regions to associate with the back side of PP2Ac and modulate phosphatase activity.     

Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms.

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.     

Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity

Sphingolipids are structural components of the eukaryotic plasma membrane that are involved, together with cholesterol, in the formation of lipid microdomains (rafts). Additionally, sphingolipid metabolites have been shown to modulate a wide variety of cellular events, including differentiation and apoptosis. To investigate the role of de novo sphingolipid biosynthesis in Leishmania, we have focused on serine palmitoyltransferase (SPT), which catalyses the first, rate-limiting step in the synthetic pathway. Genetic ablation of one SPT subunit, LmLCB2, yields viable null parasites that can no longer synthesize ceramide and sphingolipids de novo. Unexpectedly, LmLCB2 expression (and sphingolipid biosynthesis) is stage regulated in Leishmania, being undetectable in intramacrophage parasites. As expected from this observation, the LmLCB2 null mutants maintain infectivity in vivo. However, they are compromised in their ability to form infective extracellular parasites, correlating with a defect in association of the virulence factor, leishmanolysin or GP63, with lipid rafts during exocytosis and an observed relocalization of a second virulence factor, lipophosphogycan, during differentiation. Thus, de novo sphingolipid biosynthesis is critical for membrane trafficking events in extracellular Leishmania but has at best a minor role in intracellular pathogenesis.     

The Saccharomyces Cerevisiae Spt8 Gene Encodes a Very Acidic Protein That Is Functionally Related to Spt3 and Tata-Binding Protein

Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the β subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.     

Identification and characterization of an alternatively spliced isoform of the human protein phosphatase 2Aα catalytic subunit

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.     

Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques

Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.