Evidence for HTR1A and LHPP as interacting genetic risk factors in major depression.

The HTR1A -1019C>G genotype was associated with major depression in the Utah population. Linkage analysis on Utah pedigrees with strong family histories of major depression including only cases with the HTR1A -1019G allele revealed a linkage peak on chromosome 10 (maximum HLOD=4.4). Sequencing of all known genes in the linkage region revealed disease-segregating single-nucleotide polymorphisms (SNPs) in LHPP. LHPP SNPs were also associated with major depression in both Utah and Ashkenazi populations. Consistent with the linkage evidence, LHPP associations depended on HTR1A genotype. Lhpp or a product of a collinear brain-specific transcript, therefore, may interact with Htr1a in the pathogenesis of major depression.

Phylogenomic analysis and evolution of the potassium channel gene family.

Potassium channels govern the permeability of cells to potassium ions, thereby controlling the membrane potential. In metazoa, potassium channels are encoded by a large, diverse gene family. Previous analyses of this gene family have focused on its diversity in mammals. Here we have pursued a more comprehensive study in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes. The investigation revealed 164 potassium channel encoding genes in C. elegans, D. melanogaster, and mammals, classified into seven conserved families, which we applied to phylogenetic analysis. The trees are discussed in relation to the assignment of orthologous relationships between genes and vertebrate genome duplication.

Inhibition by penem of processing peptidases from cyanobacteria and chloroplast thylakoids.

Proteins targeted to the thylakoid lumen of plants and cyanobacteria and the periplasmic space of cyanobacteria are synthesised with N-terminal presequences which are removed following translocation across the membrane. These presequences are thought to direct translocation of the preprotein by a sec-type pathway. Detergent extracts of cyanobacterial and chloroplast membranes contain enzymes which are capable of processing precursors to the mature size. We show that the processing of a range of precursors by both cyanobacterial and chloroplast enzymes is inhibited by the penem SB216357. This is the first report of an inhibitor of these enzymes and indicates that they are type 1 signal peptidases.

Some characteristics of cytochrome f in the cyanobacterium Phormidium laminosum: its sequence and charge properties in the reaction with plastocyanin.

Part of the petCA operon was cloned and the sequence of the cytochrome f gene from the moderately thermophilic cyanobacterium Phormidium laminosum determined. A partial sequence of the petC gene encoding the Rieske iron-sulphur protein was also obtained. The cytochrome f gene encodes a mature protein of 385 residues and a leader sequence of 45 residues. The mature protein contains several acidic or neutral residues corresponding to basic residues in the turnip protein. Some of the latter are thought to be important for the interaction with plastocyanin via its "eastern' face. Many of the corresponding residues on the eastern face of P. laminosum plastocyanin are either basic or neutral instead of acidic. These comparisons suggested that the local charges on P. laminosum cytochrome f that are important for its interaction with the homologous plastocyanin may be negative rather than positive. The importance of acidic groups was confirmed by measuring the rates of reduction of horse heart cytochrome c and P. laminosum and spinach plastocyanins by the cytochrome bf complex isolated from P. laminosum. P. laminosum plastocyanin gave the highest rates, which decreased at high ionic strength, confirming the importance of positive local charges on this protein. When extrapolated to infinite ionic strength the rates observed with the two kinds of plastocyanin were similar, but cytochrome c became unreactive. An optimum was observed in the ionic strength response with P. laminosum plastocyanin.

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle.

Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane. To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particlelike chaperone Ffh. We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species. We have cloned and sequenced ffh from Synechococcus sp. PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts.

The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle.

Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane. To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particle-like chaperone Ffh. We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species. We have cloned and sequences ffh from Synechococcus sp. PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts.

Cloning and sequence analysis of a signal peptidase I from the thermophilic cyanobacterium Phormidium laminosum.

Type I signal peptidases are a widespread family of enzymes which remove the presequences from proteins translocated across cell membranes, including thylakoid and cytoplasmic membranes of cyanobacteria and thylakoid membranes of chloroplasts. We have cloned and sequenced a signal peptidase gene from the thermophilic cyanobacterium Phormidium laminosum which is believed to encode an enzyme common to both membrane systems. The deduced amino acid sequence is 203 residues long and although the overall similarity among signal peptidases is rather low there are a number of identifiable conserved regions present. The P. laminosum enzyme is predicted to have a single transmembrane domain, in contrast to other Gram-negative bacterial sequences, but similar to other type I signal peptidases.

The extrinsic proteins of photosystem II in photosynthetic organisms: distribution, properties and evolutionary implications.

Photosystem II from higher plant chloroplasts contains extrinsic proteins of molecular weights 33, 23 and 16 kDa. Cyanobacteria, which are thought to be closely related to the evolutionary progenitor of chloroplasts, have a protein homologous to the largest of these, but lack homologues of the smaller ones. Here we report immunological evidence for the occurrence of the 33 kDa extrinsic protein in red and chromophyte algae and its absence from anoxygenic photosynthetic bacteria. The 23 kDa protein was absent from all three groups. The red algal protein was found to be hydrophilic and rather more loosely associated with the thylakoid membrane than its higher plant equivalent. These findings support an origin for all plastids among the oxygenic prokaryotes, in contrast to the phylogeny inferred from ribulose bisphosphate carboxylase-oxygenase sequence data.

Components of the protein translocation machinery in the thermophilic cyanobacterium Phormidium laminosum.

Cyanobacteria possess cytoplasmic membranes, which are common to all eubacteria, and an additional internal network of thylakoid membranes. We have investigated the distribution of the protein translocation machinery between the two membrane systems. We have demonstrated that leader peptidase activity exists in both membrane systems, suggesting that newly synthesized protein precursors are targeted directly rather than sorted after translocation. Southern blot analysis suggests that the leader peptidase is likely to be a single enzyme common to both membrane systems. We have also shown that cyanobacteria possess a single SecA homologue. These results indicate that the protein translocation machinery in the two membrane systems is likely to be the same and leaves open the question of what mechanism enables the cell to sort proteins between them.

Carboxyl-terminal processing of the D1 protein and photoactivation of water-splitting in photosystem II. Partial purification and characterization of the processing enzyme from Scenedesmus obliquus and Pisum sativum.

The D1 polypeptide of photosystem II (PSII) is synthesized as a precursor that is processed by cleavage at the carboxyl terminus during assembly of the active PSII complex. A mutant of the green alga Scenedesmus obliquus, LF-1, inactive in water-splitting, lacks the D1 processing activity but assembles otherwise normal PSII complexes containing the precursor D1 molecule. We have isolated and partially purified a soluble protease from sonicated thylakoids of both wild-type S. obliquus and Pisum sativum which will process the precursor D1 molecule in PSII-enriched membranes from the LF-1 mutant to the mature size. After processing (but not before), photoactivation of these PSII membranes in the presence of manganese restores water-splitting to levels seen after photoactivation of PSII membranes from dark-grown, wild-type, cells. The protease is unable to process D1 in intact thylakoids from the LF-1 mutant but processes D1 if present during sonication of the thylakoids, indicating that processing of the carboxyl-terminal extension of D1 occurs in the lumen of the thylakoid. The processing protease from both S. obliquus and P. sativum is a single subunit enzyme of native molecular mass 33-35 kDa. Processing rate is optimal at pH 6.5. Processing in vitro is evident within 5 min and is markedly inhibited by millimolar concentrations of divalent cations (Cu, Zn greater than Mn greater than Ca, Mg) but not by any known inhibitors of the major classes of proteases. The protease is inactive against the precursors of other thylakoidal proteins and is thus distinct from the thylakoidal amino-terminal processing enzyme involved in the removal of transit peptides from cytoplasmically-synthesised proteins imported into the thylakoid lumen.