Syringomycin action gene SYR2 is essential for sphingolipid 4-hydroxylation in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.

The contryphans, a D-tryptophan-containing family of Conus peptides: interconversion between conformers.

We previously characterized contryphan-R, a D-tryptophan-containing octapeptide from the venom of Conus radiatus. In this study, we present evidence that the contryphan family of peptides is widely distributed in venoms of the fish-hunting cone snails. We purified, synthesized and characterized contryphan-Sm from Conus stercusmuscarum venom, and obtained molecular evidence for the existence of a third peptide, contryphan-P from Conus purpurascens venom ducts. The sequences of these three contryphans showed identity in seven of eight amino acids and a conserved pattern of post-translational modification. We also demonstrate that contryphan-Sm equilibrates between two distinct conformational states.

kappa-Conotoxin PVIIA is a peptide inhibiting the shaker K+ channel.

kappa-Conotoxin PVIIA (kappa-PVIIA), a 27-amino acid toxin from Conus purpurascens venom that inhibits the Shaker potassium channel, was chemically synthesized in a biologically active form. The disulfide connectivity of the peptide was determined. kappa-Conotoxin PVIIA has the following structure. This is the first Conus peptide known to target K+ channels. [structure: see text] Although the Shaker K+ channel is sensitive to kappa-PVIIA, the rat brain Kv1.1 subtype is resistant. Chimeras between Shaker and the Kv1.1 K+ channels were constructed and expressed in Xenopus oocytes. Only channels containing the putative pore-forming region between the fifth and sixth transmembrane domains of Shaker retained toxin sensitivity, indicating that the toxin target site is in this region of the channel. Evidence is presented that kappa-PVIIA interacts with the external tetraethyl-ammonium binding site on the Shaker channel. Although both kappa-PVIIA and charybdotoxin inhibit the Shaker channel, they must interact differently. The F425G Shaker mutation increases charybdotoxin affinity by 3 orders of magnitude but abolishes kappa-PVIIA sensitivity. The precursor sequence of kappa-PVIIA was deduced from a cDNA clone, revealing a prepropeptide comprising 72 amino acids. The N-terminal region of the kappa-PVIIA prepropeptide exhibits striking homology to the omega-, muO-, and delta-conotoxins. Thus, at least four pharmacologically distinct superfamilies of Conus peptides belong to the same "O" superfamily, with the omega- and kappa-conotoxins forming one branch, and the delta- and muO-conotoxins forming a second major branch.

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom.

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).

MutS mediates heteroduplex loop formation by a translocation mechanism.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Strategy for rapid immobilization of prey by a fish-hunting marine snail.

Some venomous animals capture prey with remarkable efficiency and speed. The purple cone, Conus purpurascens, uses two parallel physiological mechanisms requiring multiple neurotoxins to immobilize fish rapidly: neuromuscular block, and excitotoxic shock. The latter requires the newly characterized peptide kappa-conotoxin PVIIA, which inhibits the Shaker potassium channel 2-4, and beta-conotoxin PVIA5, which delays sodium-channel inactivation. Despite the extreme biochemical diversity in venoms, the number of effective strategic alternatives for prey capture are limited. How securely prey is initially tethered may strongly influence the venom strategy evolved by a predator.

A new family of Conus peptides targeted to the nicotinic acetylcholine receptor.

In this work, a new family of Conus peptides, the alpha A-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, alpha A-conotoxin PIVA, is as follows: [formula: see text] where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, [Pro13]alpha A-conotoxin PIVA and [Pro7,13]alpha A-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with alpha-bungarotoxin demonstrate that alpha A-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

Purification, characterization, synthesis, and cloning of the lockjaw peptide from Conus purpurascens venom.

The major groups of Conus peptides previously characterized from fish-hunting cone snail venoms (the alpha-, mu-, and omega-conotoxins) all blocked neuromuscular transmission. A novel activity, the "lockjaw peptide", from the fish-hunting Conus purpurascens, caused a rigid (instead of flaccid) paralysis in fish and increased excitability at the neuromuscular junction (instead of a block). We report the purification, biological activity, biochemical and preliminary physiological characterization, and chemical synthesis of the lockjaw peptide and the sequence of a cDNA clone encoding its precursor. Taken together, the data lead us to conclude that the lockjaw peptide is a vertebrate-specific delta-conotoxin, which targets voltage-sensitive sodium channels. The sequence of the peptide, which we designate delta-conotoxin PVIA, is (O = 4-trans-hydroxyproline) EACYAOGTFCGIKOGLCCSEFCLPGVCFG-NH2. This is the first of a diverse spectrum of Conus peptides which are excitotoxins in vertebrate systems.

Bidirectional excision in methyl-directed mismatch repair.

Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site. Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases. Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation. When the d(GATC) sequence on the unmodified strand resides 3' to the mismatch as viewed along the shorter path, excision requires exonuclease I. Excision occurring on the alternate hemimethylated heteroduplex depends on the 5'--> 3' hydrolytic activity of exonuclease VII. Coupled with the previous demonstration that repair initiates via the mismatch-provoked, MutHLS-dependent incision of the unmethylated strand at a d(GATC) sequence (Au, K.G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148), these findings indicate an excision mechanism in which helicase II displacement renders the incised strand sensitive to the appropriate single-strand exonuclease. Our data imply that hydrolysis commences at the d(GATC) site, proceeds to a point beyond the mismatch, and terminates at a number of discrete sites within a 100-nucleotide region just beyond this site. The extent of excision is therefore controlled by one or more components of the repair system.

Isolation and characterization of the Escherichia coli mutL gene product.

The Escherichia coli mutL gene product has been purified to near homogeneity from an overproducing clone. The mutL locus encodes a polypeptide of 70,000 daltons as determined by denaturing gel electrophoresis. The native molecular weight of MutL protein as calculated from the sedimentation coefficient of 5.5 S and Stokes radius of 61 A is 139,000 daltons, indicating that MutL exists as a dimer in solution. In addition to its ability to complement methyl-directed DNA mismatch repair in mutL-deficient cell-free extracts, DNase I protection experiments demonstrate that the purified MutL protein interacts with the MutS-heteroduplex DNA complex in the presence of ATP.

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.