Hydrogen-deuterium exchange mass spectrometry reveals the interaction of Fenna-Matthews-Olson protein and chlorosome CsmA protein

In green-sulfur bacterial photosynthesis, excitation energy absorbed by a peripheral antenna structure known as the chlorosome is sequentially transferred through a baseplate protein to the Fenna-Matthews-Olson (FMO) antenna protein and into the reaction center, which is embedded in the cytoplasmic membrane. The molecular details of the optimized photosystem architecture required for efficient energy transfer are only partially understood. We address here the question of how the baseplate interacts with the FMO protein by applying hydrogen/deuterium exchange coupled with enzymatic digestion and mass spectrometry analysis to reveal the binding interface of the FMO antenna protein and the CsmA baseplate protein. Several regions on the FMO protein, represented by peptides consisting of 123-129, 140-149, 150-162, 191-208, and 224-232, show significant decreases of deuterium uptake after CsmA binding. The results indicate that the CsmA protein interacts with the Bchl a #1 side of the FMO protein. A global picture including peptide-level details for the architecture of the photosystem from green-sulfur bacteria can now be drawn.     

The three-dimensional structure of CsmA: a small antenna protein from the green sulfur bacterium Chlorobium tepidum

The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly alpha-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal alpha-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna-Matthews-Olson antenna protein.     

A reconstituted light-harvesting complex from the green sulfur bacterium Chlorobium tepidum containing CsmA and bacteriochlorophyll a

Green sulfur bacteria possess two light-harvesting antenna systems, the chlorosome and the Fenna-Matthews-Olson (FMO) protein. In addition to self-aggregated bacteriochlorophyll (BChl) c, chlorosomes of Chlorobium tepidum contain a small amount of BChl a (ratio 100:1). The chlorosomal BChl a is associated with CsmA, a 6.2 kDa protein that accounts for more than 50% of the protein content of chlorosomes. This CsmA-BChl a complex is located in the chlorosome baseplate with the hydrophilic C-terminal part of CsmA in contact with the FMO protein. CsmA was purified from Chl. tepidum. Isolated chlorosomes were lyophilized and extracted with chloroform/methanol (1:1, v/v). The extract was further purified using gel filtration and reverse-phase HPLC and the purity of the preparation confirmed by SDS-PAGE. Mass spectrometric analysis showed an m/z of 6154.8, in agreement with the calculated mass of the csmA gene product after C-terminal processing. CD spectroscopy of the isolated protein showed that the main structural motif was an alpha-helix. We have reconstituted the isolated CsmA protein with BChl a in micelles of n-octyl beta-d-glucopyranoside. The resulting preparation reproduced the spectral characteristics of the CsmA-BChl a complex present in the chlorosome baseplate.     

Characterization of the csmD and csmE genes from Chlorobium tepidum. The CsmA, CsmC, CsmD, and CsmE proteins are components of the chlorosome envelope

The csmD and csmE genes, encoding two proteins of the chlorosome envelope, have been cloned and sequenced from the green sulfur bacterium Chlorobium tepidum. The csmD gene predicts a hydrophobic protein of 113 amino acids with a molecular mass of 11.1 kDa. The csmE gene was identified immediately upstream from csmD; the csmE gene predicts a protein of 82 amino acids (9.0 kDa) which is 49% identical to CsmA (Chung et al. (1994) Photosynthesis Res 41: 261–275). The CsmE protein is post-translationally processed, most likely in a manner similar to CsmA. The csmE and csmD genes are cotranscribed as a dicistronic mRNA but can also be cotranscribed with an open reading frame upstream from csmE that predicts a protein with sequence similarity to the CheY and SpoOF subclass of regulatory proteins. The CsmA, CsmC, CsmD, and CsmE proteins were overproduced in Escherichia coli, purified, and used to raise polyclonal antibodies in rabbits. Protease susceptibility mapping and agglutination experiments using these antibodies indicate that all four proteins are exposed at the surface of isolated chlorosomes and hence are probably components of the chlorosome envelope. Additionally, antigalactose antibodies were used to confirm that the galactosyl moiety of monogalactosyl diglycerol is exposed at the chlorosome surface; this is consistent with the notion that these lipids are components of the chlorosome envelope.Abbreviations BChl- bacteriochlorophyll - bp- basepair(s) - C.- Chloroflexus - Cb.- Chlorobium - Csm- chlorosome protein - csm gene encoding a chlorosome protein - IPTG isopropyl--d-thiogalatoside - nt nucleotide - ORF open reading frame - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Chlorosome Proteins Studied by MALDI-TOF-MS: Topology of CsmA in Chlorobium tepidum

Chlorosomes, the light-harvesting apparatus of green bacteria, are a unique antenna system, in which pigments are organized in aggregates rather than associated with proteins. Isolated chlorosomes from the green sulphur bacterium Chlorobium tepidum contain 10 surface-exposed proteins. Treatment of chlorosomes from Chlorobium tepidum with protease caused changes in the spectral properties of bacteriochlorophyll c and digestion of chlorosome proteins. Using SDS-PAGE analysis, immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) we have investigated the topology of the 59-residue CsmA protein. Our results show that at the N-terminus, the only amino acid available for protease degradation is the methionine. At the C-terminus, amino acids can be removed by protease treatment to produce a residual protein containing at least the sequence between residues 2 and 38. These results indicate that the N-terminal portion of the CsmA protein, which is predicted to be mainly hydrophobic, is buried in the chlorosome envelope.     

A model of the protein–pigment baseplate complex in chlorosomes of photosynthetic green bacteria

In contrast to photosynthetic reaction centers, which share the same structural architecture, more variety is found in the light-harvesting antenna systems of phototrophic organisms. The largest antenna system described, so far, is the chlorosome found in anoxygenic green bacteria, as well as in a recently discovered aerobic phototroph. Chlorosomes are the only antenna system, in which the major light-harvesting pigments are organized in self-assembled supramolecular aggregates rather than on protein scaffolds. This unique feature is believed to explain why some green bacteria are able to carry out photosynthesis at very low light intensities. Encasing the chlorosome pigments is a protein-lipid monolayer including an additional antenna complex: the baseplate, a two-dimensional paracrystalline structure containing the chlorosome protein CsmA and bacteriochlorophyll a (BChl a). In this article, we review current knowledge of the baseplate antenna complex, which physically and functionally connects the chlorosome pigments to the reaction centers via the Fenna–Matthews–Olson protein, with special emphasis on the well-studied green sulfur bacterium Chlorobaculum tepidum (previously Chlorobium tepidum). A possible role for the baseplate in the biogenesis of chlorosomes is discussed. In the final part, we present a structural model of the baseplate through combination of a recent NMR structure of CsmA and simulation of circular dichroism and optical spectra for the CsmA–BChl a complex.     

Phylogeny of the PscB reaction center protein from green sulfur bacteria

The reaction center (RC) of green sulfur bacteria has iron—sulfur clusters as terminal acceptors and is related to the Type I RC found in Heliobacter sp. and in Photosystem I (PS I) of green plants and cyanobacteria. Degenerate primers were used to retrieve the genes coding for one of the RC proteins, PscB, from 11 strains of green sulfur bacteria. PCR using the same primers gave no product with a second group of strains and the protein from these strains did not crossreact with antibodies raised against purified PscB from the first group, suggesting the presence of a high degree of variability. The sequences shared a high degree of similarity in the region coding for the binding motif for the 4Fe–4S centers. However, the N-terminal portion of the deduced protein sequences was highly variable and contained a highly positively charged, low-complexity region with repeated tetrapeptides with two alanines flanked by proline or lysine. The PscB sequences obtained fell into two major groups, and the results suggested a lack of correlation between the pigmentation of the chlorosome antenna system and the reaction center protein. There is also a lack of correlation between the grouping of the pscB sequences and the phylogeny deduced from 16S rRNA.This revised version was published online in October 2005 with corrections to the Cover Date.     

Molecular contacts for chlorosome envelope proteins revealed by cross-linking studies with chlorosomes from Chlorobium tepidum

Chlorosomes are unique light-harvesting antennae found in two phyla of green bacteria: Chlorobi and Chloroflexi. In the green sulfur bacterium Chlorobium tepidum, 10 proteins (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) exist in the chlorosome envelope. Chlorosomes from the wild type and mutants lacking a single chlorosome protein were cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and analyzed by gel electrophoresis. Similar cross-linking products were observed when the time and temperature were varied or when EDC was replaced with glutaraldehyde. Specific interactions between chlorosome proteins in cross-linked products were identified by immunoblotting with polyclonal antibodies raised against recombinant chlorosome proteins. We confirmed these interactions by demonstrating that these products were missing in appropriate mutants. Confirming the location of CsmA in the paracrystalline baseplate, cross-linking showed that CsmA forms dimers, trimers, and homomultimers as large as dodecamers and that CsmA directly interacts with the Fenna-Matthews-Olson protein. Cross-linking further suggests that the precursor form of CsmA is inserted near the edges of the baseplate, where CsmA and pre-CsmA interact with CsmB and CsmF. Several chlorosome proteins, including CsmA, CsmC, CsmD, CsmH, CsmI, CsmJ, and CsmX, were shown to exist as homomultimers in the chlorosome envelope. On the basis of the structural information obtained from these cross-linking experiments, a model for the locations and interactions of the proteins of the chlorosome envelope is proposed.     

Nine mutants of Chlorobium tepidum each unable to synthesize a different chlorosome protein still assemble functional chlorosomes

Chlorosomes of the green sulfur bacterium Chlorobium tepidum comprise mostly bacteriochlorophyll c (BChl c), small amounts of BChl a, carotenoids, and quinones surrounded by a lipid-protein envelope. These structures contain 10 different protein species (CsmA, CsmB, CsmC, CsmD, CsmE, CsmF, CsmH, CsmI, CsmJ, and CsmX) but contain relatively little total protein compared to other photosynthetic antenna complexes. Except for CsmA, which has been suggested to bind BChl a, the functions of the chlorosome proteins are not known. Nine mutants in which a single csm gene was inactivated were created; these mutants included genes encoding all chlorosome proteins except CsmA. All mutants had BChl c contents similar to that of the wild-type strain and had growth rates indistinguishable from or within approximately 90% (CsmC(-) and CsmJ(-)) of those of the wild-type strain. Chlorosomes isolated from the mutants lacked only the protein whose gene had been inactivated and were generally similar to those from the wild-type strain with respect to size, shape, and BChl c, BChl a, and carotenoid contents. However, chlorosomes from the csmC mutant were about 25% shorter than those from the wild-type strain, and the BChl c absorbance maximum was blue-shifted about 8 nm, indicating that the structure of the BChl c aggregates in these chlorosomes is altered. The results of the present study establish that, except with CsmA, when the known chlorosome proteins are eliminated individually, none of them are essential for the biogenesis, light harvesting, or structural organization of BChl c and BChl a within the chlorosome. These results demonstrate that chlorosomes are remarkably robust structures that can tolerate considerable changes in protein composition.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus. III. Energy transfer in whole cells

The transfer of excitation energy in intact cells of the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus was studied both at low temperature and under more physiological conditions. Analysis of excitation spectra measured at 4K indicates that the minor fraction of bacteriochlorophyll a present in the chlorosome functions as an intermediate in energy transfer between the main light-harvesting pigment BChl c and the membrane-bound B808-866 antenna complex. This supports the hypothesis that BChl a is associated with the base plate which connects the chlorosome with the membrane. The overall efficiency for energy transfer from the chlorosome to the membrane is only 15% at 4K. High efficiencies of close to 100% are observed above 40°C near the temperature where the cultures are grown. Cooling to 20°C resulted in a sudden drop of the transfer efficiency which appeared to originate in the chlorosome. This decrease may be related to a lipid phase transition. Further cooling mainly affected the efficiency of transfer between the chlorosome and the membrane. This effect can only partially be explained by a decreased Förster overlap between the chlorosomal BChl a and BChl a 808 associated with the membrane-bound antenna system. The temperature dependence of the fluorescence yield of BChl a 866 also appeared to be affected by lipid phase transitions, suggesting that this fluorescence can be used as a native probe of the physical state of the membrane.     

Computational determination of the pigment binding motif in the chlorosome protein a of green sulfur bacteria

We present a molecular-scale model of Bacteriochlorophyll a (BChl a) binding to the chlorosome protein A (CsmA) of Chlorobaculum tepidum, and the aggregated pigment–protein dimer, as determined from protein–ligand docking and quantum chemistry calculations. Our calculations provide strong evidence that the BChl a molecule is coordinated to the His25 residue of CsmA, with the magnesium center of the bacteriochlorin ring situated <3 Å from the imidazole nitrogen atom of the histidine sidechain, and the phytyl tail aligned along the nonpolar residues of the α-helix of CsmA. We also confirm that the Q _y band in the absorption spectra of BChl a experiences a large (+16 to +43 nm) redshift when aggregated with another BChl a molecule in the CsmA dimer, compared to the BChl a in solvent; this redshift has been previously established by experimental researchers. We propose that our model of the BChl a–CsmA binding motif, where the dimer contains parallel aligned N-terminal regions, serves as the smallest repeating unit in a larger model of the para-crystalline chlorosome baseplate protein.     

A comparative study of the optical characteristics of intact cells of photosynthetic green sulfur bacteria containing bacteriochlorophyll c,d or e

Energy transfer and pigment arrangement in intact cells of the green sulfur bacteria Prosthecochloris aestuarii, Chlorobium vibrioforme and chlorobium phaeovibrioides, containing bacteriochlorophyll (BChl) c, d or e as main light harvesting pigment, respectively, were studied by means of absorption, fluorescence, circular dichroism and linear dichroism spectroscopy at low temperature. The results indicate a very similar composition of the antenna in the three species and a very similar structure of main light harvesting components, the chlorosome and the membrane-bound BChl a protein. In all three species the Q_y transition dipoles of BChl c, d or e are oriented approximately parallel to the long axis of the chlorosome. Absorption and fluorescence excitation spectra demonstrate the presence of at least two BChl c-e pools in the chlorosomes of all three species, long-wavelength absorbing BChls being closest to the membrane. In C. phaeovibrioides, energy from BChl e is transferred with an efficiency of 25% to the chlorosomal BChl a at 6 K, whereas the efficiency of transfer from BChl e to the BChl a protein is 10%. These numbers are compatible with the hypothesis that the chlorosomal BChl a is an intermediary in the energy transfer from the chlorosome to the membrane.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - CD circular dichroism - LD linear dichroism     

Cryo-electron microscopy structure of the intact photosynthetic light-harvesting antenna-reaction center complex from a green sulfur bacterium

The photosynthetic reaction center complex (RCC) of green sulfur bacteria (GSB) consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna–Matthews–Olson (FMO). Functionally, FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs. In vivo, one RC was found to bind two FMOs, however, the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified. Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9 Å resolution by cryo-electron microscopy. The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously. We also observed two new subunits (PscE and PscF) and the N-terminal transmembrane domain of a cytochrome-containing subunit (PscC) in the structure. These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex. A new bacteriochlorophyll (numbered as 816) was identified at the interspace between PscF and PscA-1, causing an asymmetrical energy transfer from the two FMO trimers to RC core. Based on the structure, we propose an energy transfer network within this photosynthetic apparatus.     

Characterization of the chlorosome antenna of the filamentous anoxygenic phototrophic bacterium<Emphasis Type="Italic"> Chloronema</Emphasis> sp. strain UdG9001

The absorption and fluorescence properties of chlorosomes of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001 were analyzed. The chlorosome antenna of Chloronema consists of bacteriochlorophyll (BChl) d and BChl c together with -carotene as the main carotenoid. HPLC analysis combined with APCI LC-MS/MS showed that the chlorosomal BChls comprise a highly diverse array of homologues that differ in both the degree of alkylation of the macrocycle at C-8 and/or C-12 and the alcohol moiety esterified to the propionic acid group at C-17. BChl c and BChl d from Chloronema were mainly esterified with geranylgeraniol (33% of the total), heptadecanol (24%), octadecenol (19%), octadecanol (14%), and hexadecenol (9%). Despite this pigment heterogeneity, fluorescence emission of the chlorosomes showed a single peak centered at 765 nm upon excitation at wavelengths ranging from 710 to 740 nm. This single emission, assigned to BChl c, indicates an energy transfer from BChl d to BChl c within the same chlorosome. Likewise, incubation of chlorosomes under reducing conditions caused a weak increase in fluorescence emission, which indicates a small redox-dependent fluorescence. Finally, protein analysis of Chloronema chlorosomes using SDS-PAGE and MALDI-TOF-MS revealed the presence of a chlorosomal polypeptide with a molecular mass of 5.7 kDa, resembling the CsmA protein found in Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. Several minor polypeptides were also detected but not identified. These results indicate that, compared with other members of filamentous anoxygenic phototrophic bacteria and green sulfur bacteria, Chloronema possesses an antenna system with novel features that may be of interest for further investigations.Abbreviations APCI LC-MS/MS Atmospheric pressure chemical ionization liquid chromatography mass spectrometry - BChl Bacteriochlorophyll - Chl. Chlorobium - Cfl. Chloroflexus - MALDI-TOF-MS Matrix assisted laser desorption/ionization time-of-flight mass spectrometry - [Et] Ethyl - [i-Bu] Isobutyl - [Me] Methyl - [neo-Pent] Neopentyl - [n-Pr] Propyl - t _R Retention time     

Biosynthesis of chlorosome proteins is not inhibited in acetylene-treated cultures of Chlorobium vibrioforme

The composition, abundance and apparent molecular masses of chlorosome polypeptides from Chlorobium tepidum and Chlorobium vibrioforme 8327 were compared. The most abundant, low-molecular-mass chlorosome polypeptides of both strains had similar electrophoretic mobilities and abundances, but several of the larger proteins were different in both apparent mass and abundance. Polyclonal antisera raised against recombinant chlorosome proteins of Cb. tepidum recognized the homologous proteins in Cb. vibrioforme, and a one-to-one correspondence between the chlorosome proteins of the two species was confirmed. As previously shown [Ormerod et al. (1990) J Bacteriol 172: 1352–1360], acetylene strongly suppressed the synthesis of bacteriochlorophyll c in Cb. vibrioforme strain 8327. No correlation was found between the bacteriochlorophyll c content of cells and the cellular content of chlorosome proteins. Nine of ten chlorosome proteins were detected in acetylene-treated cultures, and the chlorosome proteins were generally present in similar amounts in control and acetylene-treated cells. These results suggest that the synthesis of chlorosome proteins and the assembly of the chlorosome envelope is constitutive. It remains possible that the synthesis of bacteriochlorophyll c and its insertion into chlorosomes might be regulated by environmental parameters such as light intensity.This revised version was published online in October 2005 with corrections to the Cover Date.     

The three-dimensional structure of the FMO protein from <Emphasis Type="Italic">Pelodictyon phaeum</Emphasis> and the implications for energy transfer

The Fenna–Matthews–Olson (FMO) antenna protein from the green bacterium Pelodictyon phaeum mediates the transfer of energy from the peripheral chlorosome antenna complex to the membrane-bound reaction center. The three-dimensional structure of this protein has been solved using protein crystallography to a resolution limit of 2.0 Å, with R _work and R _free values of 16.6 and 19.9%, respectively. The structure is a trimer of three identical subunits related by a threefold symmetry axis. Each subunit has two beta sheets that surround 8 bacteriochlorophylls. The bacteriochlorophylls are all five-coordinated, with the axial ligand being a histidine, serine, backbone carbonyl, or bound water molecule. The arrangement of the bacteriochlorophylls is generally well conserved in comparison to other FMO structures, but differences are apparent in the interactions with the surrounding protein. In this structure the position and orientation of the eighth bacteriochlorophyll is well defined and shows differences in its location and the coordination of the central Mg compared to previous models. The implications of this structure on the ability of the FMO protein to perform energy transfer are discussed in terms of the experimental optical measurements.     

Sequence, expression and localization of the immunity protein for colicin B

Summary Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20 185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.     

Selective protein extraction from Chlorobium tepidum chlorosomes using detergents. Evidence that CsmA forms multimers and binds bacteriochlorophyll a

Chlorosomes of the photosynthetic green sulfur bacterium Chlorobium tepidum consist of bacteriochlorophyll (BChl) c aggregates that are surrounded by a lipid-protein monolayer envelope that contains ten different proteins. Chlorosomes also contain a small amount of BChl a, but the organization and location of this BChl a are not yet clearly understood. Chlorosomes were treated with sodium dodecyl sulfate (SDS), Lubrol PX, or Triton X-100, separately or in combination with 1-hexanol, and the extracted components were separated from the residual chlorosomes by ultrafiltration on centrifugal filters. When chlorosomes were treated with low concentrations of SDS, all proteins except CsmA were extracted. However, this treatment did not significantly alter the size and shape of the chlorosomes, did not extract the BChl a, and caused only minor changes in the absorption spectrum of the chlorosomes. Cross-linking studies with SDS-treated chlorosomes revealed the presence of multimers of the major chlorosome protein, CsmA, up to homooctamers. Extraction of chlorosomes with SDS and 1-hexanol solubilized all ten chlorosome envelope proteins as well as BChl a. Although the size and shape of these extracted chlorosomes did not initially differ significantly from untreated chlorosomes, the extracted chlorosomes gradually disintegrated, and rod-shaped BChl c aggregates were sometimes observed. These results strongly suggest that CsmA binds the BChl a in Chlorobium-type chlorosomes and further indicate that none of the nine other chlorosome envelope proteins are absolutely required for maintaining the shape and integrity of chlorosomes. Quantitative estimates suggest that chlorosomes contain approximately equimolar amounts of CsmA and BChl a and that roughly one-third of the surface of the chlorosome is covered by CsmA.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein.

Summary In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing. Comparison with the published wild-type sequence from strain SKI revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds. Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain. Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange. The latter is therefore responsible for the temperature-sensitive phenotype. A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SKI, is not temperature sensitive. The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SKI is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.