Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ⁻ cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon. Received: 8 October 1996 / Accepted: 27 November 1996     

N-type Inactivation in the Mammalian Shaker K+ Channel Kv1.4

Mammalian voltage-gated K⁺ channels are oligomeric proteins, some of which may be composed in vivo of subunits derived from several similar genes. We have studied N-type inactivation in the rapidly inactivating Kv1.4 channel and, in specific, heteromultimers of this gene product with Kv1.5 noninactivating subunits. Heteromultimeric channels were analyzed for the stoichiometry of Kv1.4:Kv1.5 subunits by observing shifts in the midpoints of steady-state availability from that of homomultimeric channels. This analysis was employed to examine inactivation of heteromultimeric channels expressed in Xenopus oocytes using two model systems: by expression of a Kv1.4–Kv1.5 tandem fusion construct and by coexpression of native Kv1.4 and Kv1.5 channels across a wide relative concentration range of microinjected mRNA. Additionally, inactivation was examined in coexpression experiments of N-terminal deletion mutants of Kv1.4. We found that (i) a single inactivating subunit conferred inactivation in all hetero-multimers studied; (ii) the rate of inactivation could not be distinguished in channels containing two inactivating subunits from those containing one inactivating subunit; and (iii) large deletions in the linker region between the N-terminal inactivation region and the first membrane-spanning domain had no effect on the rate of inactivation. These data confirm the importance of the proximal N-terminal region in the inactivation of mammalian Kv1.4 channels, and suggest that the inactivation particle remains in close proximity to the permeation pathway even when the channel is in the open state. Received: 24 August 1995/Revised: 7 February 1996     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the α-subunit cloned from the guinea-pig colon (αgp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of αgp, βrat and γrat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed β adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased αgp, βrat and γrat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive αgp and the cpt-cAMP-resistant αrat-subunit showed that neither the N-terminal nor the C-terminal of α ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by αgp and βγrat by interacting with the extracellular part of the protein. Received: 19 January 2001/Revised: 27 April 2001     

The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.     

Mutations in the M4 Domain of the Torpedo californica Nicotinic Acetylcholine Receptor Alter Channel Opening and Closing

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time. Received: 27 September 1996/Revised: 28 January 1997     

Stoichiometry of the Large Conductance Bacterial Mechanosensitive Channel of E. coli. A Biochemical Study

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the `extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress. Received: 26 February 1999/Revised: 10 June 1999     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Volume-regulatory Amino Acid Release from the Protozoan Parasite Crithidia luciliae

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells. Received: 13 May 1996/Revised: 9 July 1996     

Minimum domain of the Shiga toxin A subunit required for enzymatic activity.

The minimum sequence of the enzymatic (A) subunit of Shiga toxin (STX) required for activity was investigated by introducing N-terminal and C-terminal deletions in the molecule. Enzymatic activity was assessed by using an in vitro translation system. A 253-amino-acid STX A polypeptide, which is recognized as the enzymatically active portion of the 293-amino-acid A subunit, expressed less than wild-type levels of activity. In addition, alteration of the proposed nicking site between Ala-253 and Ser-254 by site-directed mutagenesis apparently prevented proteolytic processing but had no effect on the enzymatic activity of the molecule. Therefore, deletion analysis was used to identify amino acid residue 271 as the C terminus of the enzymatically active portion of the STX A subunit. STX A polypeptides with N-terminal and C-terminal deletions were released into the periplasmic space of Escherichia coli by fusion to the signal peptide and the first 22 amino acids of Shiga-like toxin type II, a member of the STX family. Although these fusion proteins expressed less than wild-type levels of enzymatic activity, they confirmed the previous finding that Tyr-77 is an active-site residue. Therefore, the minimum domain of the A polypeptide which was required for the expression of enzymatic activity was defined as StxA residues 75 to 268.     

Mechanosensitive channel of Thermoplasma, the cell wall-less archaea

By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL ⁻ knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL ⁺ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels.     

Phylogenetic,Structural and Functional Characteristics of the Na-K-Cl Cotransporter Family

Summary Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane α-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggests that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom. Received: 27 July 1995/Revised: 8 November 1995     

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme,Transketolase

Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade. Received: 13 September 1995 / Accepted: 14 November 1996     

Sequence Analyses and Phylogenetic Characterization of the ZIP Family of Metal Ion Transport Proteins

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, α-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins. Received: 31 March 1997/Revised: 14 May 1998     

Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Δg _m , was initially determined. DT-AB induced a ∼10-fold lower Δg _m than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg _m , whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg _m . Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Δg _m and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel. Received: 20 June 1996/Revised: 8 November 1996     

Gene structure of a chlorophyll a/c-binding protein from a brown alga: Presence of an intron and phylogenetic implications

A Laminaria saccharina genomic library in the phage EMBL 4 was used to isolate and sequence a full-length gene encoding a fucoxanthin-chlorophyll a/c-binding protein. Contrary to diatom homologues, the coding sequence is interrupted by an intron of about 900 bp which is located in the middle of the transit peptide. The deduced amino acid sequence of the mature protein is very similar to those of related proteins from Macrocystis pyrifera (Laminariales) and, to a lesser extent, to those from diatoms and Chrysophyceae. Seven of the eight putative chlorophyll-binding amino acids determined in green plants are also present. Alignments of different sequences related to the light-harvesting proteins (LHC) demonstrate a structural similarity among the three transmembrane helices and suggest a unique ancestral helix preceded by two β-turns. The β-turns are conserved in front of the second helices of the chlorophyll a/c proteins more so than in chlorophyll a/b proteins. Phylogenetic trees generated from sequence data indicate that fucoxanthin-chlorophyll-binding proteins diverged prior to the separation of photosystem I and photosystem II LHC genes of green plants. Among the fucoxanthin-containing algae, LHC I or II families could not be distinguished at this time. Received: 14 February 1996 / Accepted: 4 April 1996     

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Evolutionary Comparisons of RecA-Like Proteins Across All Major Kingdoms of Living Organisms

Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively. Received: 25 October 1996 / Accepted: 31 December 1996