Membrane Targeting and Insertion of the C-Tail Protein SciP.

C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-terminal part of the SciP protein sequence are present. Two short hydrophobic sequences were identified that targeted a green fluorescent protein-SciP fusion protein to the membrane involving the signal recognition particle. After targeting, the membrane insertion of SciP is catalyzed by YidC independent of the SecYEG translocase. However, when the C-terminal tail of SciP was extended to 21 aa residues, we found that SecYEG becomes involved and makes its membrane insertion more efficient.

The T7 ejection nanomachine components gp15-gp16 form a spiral ring complex that binds DNA and a lipid membrane.

Bacteriophage T7 initiates infection by ejecting several internal capsid proteins into the host cell; these proteins then assemble into a nanomachine that translocates the viral genome from the phage head into the cytoplasm. The ejected proteins are thought to partially unfold as they pass through the lumen of the portal and the short stubby T7 tail during their entry into the cell. In vivo, the internal proteins gp15 and gp16 assemble into a tubular structure that spans the periplasm and cytoplasmic membrane. We show here that purified gp15 and gp16 can refold from a partially denatured state in vitro, and that gp15 interacts with gp16 to form a spiral ring structure. Purified gp15 binds to DNA, whereas gp16 binds protein-free liposomes; the gp15-gp16 complex binds both DNA and liposomes. Limited proteolysis of the liposome-bound gp16 reveals that its C-terminal region is protected, suggesting a partial membrane insertion of the protein.

Mdm38 is a 14-3-3-like receptor and associates with the protein synthesis machinery at the inner mitochondrial membrane.

Mitochondrial ribosomes synthesize core subunits of the inner membrane respiratory chain complexes. In mitochondria, translation is regulated by mRNA-specific activator proteins and occurs on membrane-associated ribosomes. Mdm38/Letm1 is a conserved membrane receptor for mitochondrial ribosomes and specifically involved in respiratory chain biogenesis. In addition, Mdm38 and its higher eukaryotic homolog Letm1, function as K(+)/H(+) or Ca(2+)/H(+) antiporters in the inner membrane. Here, we identify the conserved ribosome-binding domain (RBD) of Mdm38 and determine the crystal structure at 2.1 Å resolution. Surprisingly, Mdm38(RBD) displays a 14-3-3-like fold despite any similarity to 14-3-3-proteins at the primary sequence level and thus represents the first 14-3-3-like protein in mitochondria. The 14-3-3-like domain is critical for respiratory chain assembly through regulation of Cox1 and Cytb translation. We show that this function can be spatially separated from the ion transport activity of the membrane integrated portion of Mdm38. On the basis of the phenotypes observed for mdm38Δ as compared to Mdm38 lacking the RBD, we suggest a model that combining ion transport and translational regulation into one molecule allows for direct coupling of ion flux across the inner membrane, and serves as a signal for the translation of mitochondrial membrane proteins via its direct association with the protein synthesis machinery.

Excitation energy pathways in the photosynthetic units of reaction center LM- and H-subunit deletion mutants of Rhodospirillum rubrum.

Light-induced reaction dynamics of isolated photosynthetic membranes obtained from wild-type (WT) and reaction center (RC)-subunit deletion strains SPUHK1 (an H-subunit deletion mutant) and SK Delta LM (an (L+M) deletion mutant) of the purple non-sulphur bacterium Rhodospirillum rubrum have been investigated by femtosecond transient absorption spectroscopy. Upon excitation of the spirilloxanthin (Spx) S(2) state at 546 nm, of the bacteriochlorophyll Soret band at 388 nm and probing spectral regions, which are characteristic for carotenoids, similar dynamics in the SPUHK1, SK Delta LM and WT strains could be observed. The excitation of Spx S(2) is followed by the simultaneous population of the lower singlet excited states S(1) and S* which decay with lifetimes of 1.4 and 5 ps, respectively for the mutants, and 1.4 and 4 ps, respectively, for the wild-type. The excitation of the BChl Soret band is followed by relaxation into BChl lower excited states which compete with excitation energy transfer BChl-to-Spx. The deexcitation pathway BChl(Soret) --> Spx(S(2)) --> Spx(S(1)) occurs with the same transition rate for all investigated samples (WT, SPUHK1 and SK Delta LM). The kinetic traces measured for the Spx S(1) --> S(N) transition display similar behaviour for all samples showing a positive signal which increases within the first 400 fs (i.e. the time needed for the excitation energy to reach the Spx S(1) excited state) and decays with a lifetime of about 1.5 ps. This suggests that the Spx excited state dynamics in the investigated complexes do not differ significantly. Moreover, a longer excited state lifetime of BChl for SPUHK1 in comparison to WT was observed, consistent with a photochemical quenching channel present in the presence of RC. For long delay times, photobleaching of the RC special pair and an electrochromic blue shift of the monomeric BChl a can be observed only for the WT but not for the mutants. The close similarity of the excited state decay processes of all strains indicates that the pigment geometry of the LH1 complex in native membranes is unaffected by the presence of an RC and allows us to draw a model representation of the WT, SK Delta LM and SPUHK1 PSU complexes.

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB.

The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic "gate" formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH(4)(+) deprotonation takes place at S1 before NH(3) conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

The 1.3-A resolution structure of Nitrosomonas europaea Rh50 and mechanistic implications for NH3 transport by Rhesus family proteins.

The Rhesus (Rh) proteins are a family of integral membrane proteins found throughout the animal kingdom that also occur in a number of lower eukaryotes. The significance of Rh proteins derives from their presence in the human red blood cell membrane, where they constitute the second most important group of antigens used in transfusion medicine after the ABO group. Rh proteins are related to the ammonium transport (Amt) protein family and there is considerable evidence that, like Amt proteins, they function as ammonia channels. We have now solved the structure of a rare bacterial homologue (from Nitrosomonas europaea) of human Rh50 proteins at a resolution of 1.3 A. The protein is a trimer, and analysis of its subunit interface strongly argues that all Rh proteins are likely to be homotrimers and that the human erythrocyte proteins RhAG and RhCE/D are unlikely to form heterooligomers as previously proposed. When compared with structures of bacterial Amt proteins, NeRh50 shows several distinctive features of the substrate conduction pathway that support the concept that Rh proteins have much lower ammonium affinities than Amt proteins and might potentially function bidirectionally.

Reprint of "Structural and mechanistic aspects of Amt/Rh proteins" [J. Struct. Biol. 158 (2007) 472-481].

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport. The Escherichia coli channel AmtB has become the model system of choice for analysis of the mechanism of ammonia conductance, increasingly also through molecular dynamics simulations. Further progress in a more detailed mechanistic understanding of these proteins requires a reliable in vitro assay using purified protein, allowing quantitative kinetic measurements under a variety of experimental conditions for different Amt/Rh proteins, including mutants. Here, we critically review the existing functional data in the context of the most interesting and unresolved mechanistic questions and we present our results, obtained using an in vitro assay set up with the purified E. coli channel AmtB.

Structural and mechanistic aspects of Amt/Rh proteins.

Amt/Rh proteins, which mediate movement of ammonium across cell membranes, are spread throughout the three kingdoms of life. Most functional studies on various members of the family have been performed using cellular assays in heterologous expression systems, which are, however, not very well suited for detailed mechanistic studies. Although now generally considered to be ammonia conducting channels, based on a number of experimental studies and structural insights, the possibility remains that some plant Amts facilitate net ammonium ion transport. The Escherichia coli channel AmtB has become the model system of choice for analysis of the mechanism of ammonia conductance, increasingly also through molecular dynamics simulations. Further progress in a more detailed mechanistic understanding of these proteins requires a reliable in vitro assay using purified protein, allowing quantitative kinetic measurements under a variety of experimental conditions for different Amt/Rh proteins, including mutants. Here, we critically review the existing functional data in the context of the most interesting and unresolved mechanistic questions and we present our results, obtained using an in vitro assay set up with the purified E. coli channel AmtB.

The crystal structure of the Escherichia coli AmtB-GlnK complex reveals how GlnK regulates the ammonia channel.

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the PII signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.5 A. This structure of PII in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GlnK. GlnK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation.

An unusual twin-his arrangement in the pore of ammonia channels is essential for substrate conductance.

Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.

The reaction center H subunit is not required for high levels of light-harvesting complex 1 in Rhodospirillum rubrum mutants.

The gene (puhA) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center.

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of