The regulatory C-terminal domain of subunit ε of F₀F₁ ATP synthase is dispensable for growth and survival of Escherichia coli

The C-terminal domain of subunit ε of the bacterial F_oF₁ ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.F_oF₁ ATP synthases are ubiquitous enzymes that synthesize ATP using a transmembrane electrochemical potential of protons or proton motive force (PMF) generated by the respiratory chain across the cytoplasmic membrane of bacteria, the thylakoid membrane of chloroplasts, or the mitochondrial inner membrane (4, 5, 37). The enzyme consists of two parts: membrane-embedded F_o subcomplex (a complex of subunits a, b, and c in bacteria) and hydrophilic F₁ subcomplex (composed of subunits α, β, γ, δ, and ε). The enzyme is also known as a molecular motor, which is composed of the stator subcomplex (α, β, δ, a, and b) and the rotor subcomplex (γ, ε, and c), and its rotation is coupled to ATP synthesis and proton flow across the membrane (20, 31, 52). The reaction of the enzyme is reversible; ATP is hydrolyzed into ADP and inorganic phosphate, the rotor subcomplex rotates in reverse, and protons are extruded to the periplasmic side, resulting in the generation of PMF. Although some bacteria utilize the reverse reaction under particular conditions, the primary function of F_oF₁ ATP synthase is generation of ATP from the PMF. Therefore, the direction of the activity of F_oF₁ ATP synthase is regulated to avoid wasteful ATP hydrolysis.Subunit ε in bacterial F_oF₁ has been known to be an intrinsic inhibitor of F₁ and F_oF₁ complex (18, 21, 23) and is proposed to have a regulatory function (10, 11, 42). Although the inhibitory effects of subunit ε vary among species, in general, ε inhibits ATP hydrolysis activity while repressing ATP synthesis activity to a lesser degree (14, 27). This regulatory function of the ε subunit is mediated almost exclusively by the C-terminal region of ε, which is comprised of two antiparallel α-helices (18, 49, 50). Biochemical and crystallographic studies have revealed that the C-terminal helices can adopt two different conformations (34, 46, 47, 48). In the retracted conformation, the α-helices form a hairpin-like structure and sit on the N-terminal β-sandwich domain of the ε subunit. When the ε subunit exhibits an inhibitory effect, it adopts a more extended conformation in which the C-terminal α-helices extend along the γ subunit, which composes the central stalk. It has also been shown that basic, positively charged residues on the second α-helix of the ε subunit interact with negatively charged residues in the DELSEED segment of subunit β to exert the inhibitory effect (12).Escherichia coli mutants deleted in the entire ε subunit exhibit a reduced growth rate and growth yield, and this effect is proposed to be a result of a deficiency in assembly of the F_o and F₁ complexes (21). The N-terminal β-sandwich domain of the ε subunit is responsible for the assembly of F_o and F₁ and is therefore important for efficient coupling between proton translocation through F_o and ATP synthesis/hydrolysis in F₁ (15, 39). Deletion of the ε subunit leads to dissociation of the F_oF₁ complex and wasteful ATP hydrolysis by free (cytoplasmic) F₁ and dissipation of PMF through free F_o (21, 22, 51).While the importance of the entire ε subunit in the whole-cell physiology of E. coli is fairly well established, the role of the regulatory C-terminal region of ε has received little attention and warrants investigation to determine if the regulatory functions (e.g., inhibition of ATP hydrolysis) observed in vitro are manifested in the physiology of E. coli under various growth conditions. To address this question, we constructed isogenic E. coli mutants that were deleted in the C-terminal region of ε subunit (ε^DC) and used these strains to compare physiological properties of wild-type versus ε^DC cells under a wide range of environmental conditions and stressors.     

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

The structure of the membrane integral rotor ring of the proton translocating F₁F₀ ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c₁₁ rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6–10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu⁶¹ in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu⁶¹ is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu⁶¹ by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.ATP synthases found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts catalyze ATP synthesis and ATP hydrolysis coupled with transmembrane proton or sodium ion transport. The enzymes are multi-subunit complexes composed of an extra-membranous catalytic F₁ domain and an interconnected integral membrane F₀ domain. The hydrophilic F₁ domain consists of five different polypeptides with a stoichiometry of α₃β₃γδϵ. Detailed structural information obtained with the mitochondrial enzyme (1–3) in combination with biochemical (4), biophysical (5), and single molecule studies (6–9) revealed that synthesis or hydrolysis of ATP in the F₁ domain is accomplished via a rotary catalytic mechanism. In addition to information on the catalytic mechanism, structure analysis and single molecule studies of the mitochondrial or the chloroplast F₁ complex have also unraveled the molecular mechanism of several F₁-specific inhibitors (10–14). Less detailed information is available on the integral membrane F₀ domain, which consists of three different polypeptides (a, b, and c) and mediates the transfer of protons or sodium ions across the membrane. Subunits a and b were shown to reside at the periphery of a cylindrical complex formed by multiple copies of the c subunit (15–18). The number of c subunits in the cylindrical subcomplex shows substantial variation in different organisms. Ten protomers are found in ATP synthases from yeast, Escherichia coli and Bacillus PS3 (19–21), 11 in Ilyobacter tartaricus, Propionigenium modestum, and Clostridium paradoxum (22–24), 13 in the thermoalkalophilic Bacillus TA2.TA1 (25), 14 in spinach chloroplasts (26), and 15 in the cyanobacterium Spirulina platensis (27). The structure of isolated subunits a, b, and c from E. coli has been studied by mutagenesis analysis and by NMR spectroscopy in a mixed solvent that was suggested to mimic the membrane environment (28–32). These studies showed that subunit a folds with five membrane-spanning helices. The fourth of these helices directly interacts with subunit c and contains a conserved arginine (Arg²¹⁰), which is thought to be involved in proton transfer (33). Subunit b, which is present in two copies in the intact F₀, contains a single transmembrane helix. Cross-linking data support a direct interaction of the two copies of the b subunit (29). Subunit c was studied at two different pH values to obtain the protonated and deprotonated form of a conserved carboxylate (Asp⁶¹ in E. coli) that was shown to be essential for proton transport (34). NMR spectroscopy revealed that the isolated c subunit consists of two long hydrophobic membrane spanning segments connected by a short hydrophilic loop (30, 35). This loop is located close to the γ and ϵ subunit on the F₁ side of the membrane (36, 37). Low resolution x-ray crystallography, cryo-electron microscopy, and atomic force microscopy showed that the membrane-spanning helices of the multiple copies of subunit c in the intact F₀ complex are tightly packed in two concentric rings (19, 22, 26). Atomic resolution of the c ring was recently provided for the Na⁺-translocating F-type ATPase from I. tartaricus (38) and the related Na⁺-translocating V-type ATPase from Enterococcus hirae (39). Rotation of the c ring was demonstrated by cross-linking (18), fluorescence studies (40), and single molecule visualization (41, 42). Based on the structural and biochemical information on F₁ and F₀, different mechanical models have been proposed describing how the rotation of the c ring is coupled to the rotation of the F₁ rotor subunits. This rotation in turn drives sequential conformational shifts at the three catalytic β subunits that result in ATP synthesis (43–45). Vice versa hydrolysis of ATP in the F₁ domain is thought to drive rotation of the γϵc_10–15 subcomplex and transports protons or sodium ions across the membrane.Here we describe the crystal structure of the chloroplast c₁₄ rotor, which is the first structure of an isolated c ring rotor from a proton driven ATPase. The structure was solved by molecular replacement using a tetradecameric search model that was generated from a monomer taken from the I. tartaricus c₁₁ structure. The imposition of noncrystallographic symmetry restraints during refinement substantially improved electron density and structure determination.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag⁺ and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag⁺ sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag⁺ sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd²⁺, a membrane-impermeant soft metal ion with properties similar to Ag⁺. Cd²⁺ inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.F₁F₀ ATP synthase utilizes the energy stored in an H⁺ or Na⁺ electrochemical gradient to synthesize ATP in bacteria, mitochondria, and chloroplasts (1–4). The ATP synthase complex is composed of two sectors, i.e. a water-soluble F₁ sector that is bound to a membrane-embedded F₀ sector. In bacteria, F₁ is composed of five subunits in an α₃β₃γδϵ ratio and contains three catalytic sites for ATP synthesis and/or hydrolysis centered at the α-β subunit interfaces. F₀ is composed of three subunits in an a₁b₂c_10–15 ratio and functions as the ion-conducting pathway (5–9). Ion translocation through F₀ drives rotation of a cylindrical ring of c-subunits that is coupled to rotation of the γ subunit within the (αβ)₃ hexamer of F₁ to force conformational changes in the three active sites and in turn drive synthesis of ATP by the binding change mechanism (1–4, 10–13).Subunit c of F₀ folds in the membrane as a hairpin of two extended α-helices. In Escherichia coli, 10 copies of subunit c pack together to form a decameric ring with TMH1² on the inside and TMH2 on the periphery (6, 14). An atomic resolution structure of the Na⁺-translocating c₁₁-ring from Ilyobacter tartaricus was recently published by Meier et al. (8). In the c₁₁ structure, the Na⁺ binding site is formed by two interacting c subunits. The essential Na⁺-binding Glu residue, which corresponds to Asp-61 in E. coli, is located in TMH2 at the middle of the lipid bilayer. Subunit a consists of five transmembrane helices, four of which likely interact as a four-helix bundle (15–18). Subunit a lies on the periphery of the c-ring with TMHs 4 and 5 from subunit a and TMH2 from subunit c forming the a-c interface (18–21). During ion translocation through F₀, the essential Arg-210 on TMH4 of subunit a is postulated to facilitate the protonation/deprotonation cycle at Asp-61 of subunit c and cause the rotation of the c-ring past the stationary subunit a (3, 4, 19).Chemical modification of cysteine-substituted transmembrane proteins has been widely used as a means of probing the aqueous accessible regions (22–24). The reactivity of a substituted cysteine to thiolate-directed probes provides an indication of aqueous accessibility because the reactive thiolate species is preferentially formed in an aqueous environment. The aqueous accessibility of the five TMHs in subunit a of E. coli F₀ has been probed using Ag⁺ and NEM (19, 25–27). The results suggest the presence of an aqueous accessible channel in subunit a in the center of TMHs 2–5 extending from the periplasm to the center of the membrane. Protons entering through this periplasmic access channel are postulated to bind to the essential Asp-61 residues of the c-ring and exit to the cytoplasm by a still uncertain pathway at the peripheral face of aTMH4 with protonation/deprotonation of Asp-61 driving c-ring rotation.During H⁺-driven ATP synthesis, two models for the pathway by which H⁺ or Na⁺ exit to the cytoplasm have been proposed. The first model proposes that the ions bound at Asp-61 exit to the cytoplasm via a half-channel composed at least partially by residues in TMH4 of subunit a (25–27). Chemical modification studies of Cys-substituted subunit a of E. coli revealed an aqueous accessible surface of TMH4 that includes the essential Arg-210 residue, which extended from the center of the membrane to the cytoplasm, suggesting that the ion exit channel may lie at the a-c interface (19, 25). Alternatively studies of the c-ring from the I. tartaricus enzyme indicate that Na⁺ can access Glu-65 in the absence of other F₀ subunits, suggesting an intrinsic channel in subunit c (28, 29). However, no such channel was apparent in the crystal structure of the c₁₁-ring (8). In a previous study (30), we probed the thiolate reactivity of Cys substitutions in the cytoplasmic half of TMH2 in subunit c. These experiments revealed extensive reactivity to sulfhydryl-directed reagents on the peripheral face of cTMH2, supporting the presence of the cytoplasmic exit channel at the a-c interface. In this study, we extended the survey of aqueous accessibility in transmembrane regions by probing thiolate reactivity of Cys substitutions in TMH1 and in the periplasmic half of TMH2. The reactivity of Cys substituted into these regions proved to be more limited. Only a small region of TMH1, lying directly behind Asp-61, was reactive with Ag⁺. In addition to Ag⁺, we used Cd²⁺ as a complementary, membrane-impermeant probe for aqueous accessibility. The survey of Cd²⁺ sensitivity confirmed that aqueous accessibility from the cytoplasm is much greater for residues packing at the periphery of the c-ring. The experiments reported here distinguish the aqueous accessible and inaccessible regions of the c-ring and strengthen evidence that the cytoplasmic H⁺ exit channel is situated at the a-c interface.     

The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membrane-bound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V₀ subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V₁ and V₀(−d) that failed to form V₁V₀, and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V₁V₀ assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V₁V₀ was delivered to the vacuole and active. It retained 63–71% of the wild-type activity and was responsive to glucose. Tether-less V₁V₀ disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V₁ subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V₀ assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6.V-ATPase⁴ proton pumps are highly conserved proteins fundamental for pH homeostasis (for review, See Refs. 1–6). Located in the endomembrane system, V-ATPases establish and maintain the low pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting (for review, see Refs. 1–3). Cells specialized for active proton secretion, like kidney epithelial cells and osteoclasts, also express V-ATPases at the plasma membrane, where they transfer protons from the cytosol to the extracellular milieu (4, 5). In the kidney, plasma membrane V-ATPases of the intercalated cells are critical for regulation of the systemic acid-base balance (5, 6). Mutations in human kidney V-ATPase cause distal-renal tubular acidosis (6). V-ATPases at the plasma membrane of osteoclasts are essential for bone resorption, and mutations result in osteopetrosis, a disease characterized by thickening of the bones (1, 4, 7). Complete loss of V-ATPase activity is lethal in eukaryotes other than fungi (3).V-ATPases are multisubunit complexes that consist of two domains, V₁ (peripheral) and V₀ (membrane-bound) (1, 2). Each of the subunits in the V-ATPase complex is critical for function and V₁V₀ assembly (8). Deletion of a peripheral V₁ subunit leads to disruption of the entire V₁ domain in yeast. Loss of a V₀ subunit does not affect V₁ assembly but disrupts the entire V₀ domain, which also prevents V₁ from associating with the membrane. An exception is subunit a for which two functional isoforms (Vph1, Stv1) exist in yeast (9). Disruption of subunit a requires disruption of both genes (9).Eight different subunits (A-H) compose the V₁ domain where ATP hydrolysis takes place at a catalytic hexamer A₃B₃ (1). Six subunits (a, c, c′, c′′, d, e) form V₀, the membrane intrinsic domain that holds V₁ and forms the path for proton transport via a hydrophobic ring structure (c-ring). V₁ and V₀ subunits contribute to the formation of one central and three peripheral stalks that connect the c-ring and the catalytic hexamer A₃B₃ (1). ATP hydrolysis drives rotation of the central stalk (connected to the c-ring) (10). Protons are transferred from the cytosol to each subunit of the c-ring and from the c-ring to the other side of the membrane passing through subunit a (11). As many protons, as subunits forming the c-ring, get transferred against a concentration gradient when hydrolysis of three ATP molecules powers 360° rotation.V-ATPases are related to F-ATP (F₁F₀ ATP) synthases. Both proteins work as molecular motors (10, 12–14). It is postulated that the asymmetry imposed by having a 3-fold symmetry in F₁ (and V₁) and an apparent 10-fold symmetry in the c-ring of F₀ (and V₀) requires energy to be transiently stored. The energy of coupling is thought to be stored in the peripheral (stator) and central (rotor) stalk structures of F₁F₀ (15–17). Subunit a is the only peripheral stalk component of the V-ATPase complex that is secured in the membrane (18). It is key for maintaining structural stability when relative rotation of subunits occurs during catalysis. Thus, the tether of subunit a in V₀ could be functionally comparable with the tether of subunit b in F₀ (Escherichia coli), although subunits a (V₀) and b (F₀) do not share sequence homology.Subunit a is a 95-kDa protein that consists of two domains that are structurally and functionally distinguishable. The hydrophilic N-terminal domain (∼45 kDa) is oriented toward the cytosolic side of the membrane and contains the information necessary to deliver V-ATPases to different compartments (19). The N terminus interacts with multiple V₁ subunits, including the catalytic subunit A (20) and peripheral stalk-forming subunits H, C, E, and G of V₁ (18, 21). It is through these interactions that the N-terminal domain serves as a stator, which prevents rotation of the A₃B₃ hexamer during catalysis. The other half of subunit a, the C-terminal domain (∼50 kDa), is buried in the membrane by multiple transmembrane-spanning regions (9). The C-terminal domain interacts with the periphery of the c-ring (22) and contributes to the path for proton transport (11, 19) by providing access to cytosolic protons and directing their exit to the luminal side of the membrane.In contrast to its role as stator during catalysis, the N-terminal domain of subunit a is a movable element that switches positions when V₁V₀ is regulated by disassembly and reassembly in vivo (1, 2, 23, 24). Inactivation of V-ATPases by disassembly is a rapid response to glucose starvation in yeast (23, 25). In the absence of glucose the V-ATPase complex dissociates into three parts: V₁ subunit C, V₁ (without subunit C), and V₀ (23). Disassembly is reversible, and the three components reassociate immediately after glucose addition, restoring ATP hydrolysis and proton transport. As V₁V₀ disassembles and reassembles, the N-terminal domain of subunit a alternates between V₁V₀ and V₀ (26, 27). In V₁V₀ it contributes to stabilizing the stator-forming V₁ subunits (1, 18). In V₀, its role has yet to be determined.As its functional and regulatory roles emerge, it becomes clear that the cytosolic N terminus of V₀ subunit a is key for V₁V₀ activity, assembly, and regulation. In this study deletions were made at amino acids that connect the N-terminal and C-terminal domains of subunit a Vph1. Shrinking of the tether that anchors subunit a to the membrane harmed assembly of subunit d into V₀, making yeast cells sensitive to pH (vma growth phenotype). Growth defects were rescued by exogenous VMA6, the gene encoding subunit d. Remarkably, subunit d restored assembly and significant function of V-ATPase proton pumps that had up to 46 residues of the tether removed. Because V₁V₀ containing tether-less vph1 assembled with peripheral V₁ subunits and with the glycolytic enzyme phosphofructokinase, we concluded that no major structural changes were generated at the N- and C-terminal domains. Early steps of V₀ assembly, and trafficking were likely impaired by shorter tethers and rescued by VMA6. The potential mechanisms by which overexpression of subunit d rescued subunit a deletions are discussed.     

Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family

The Friend spleen focus-forming virus (SFFV) env gene encodes a glycoprotein with apparent M_r of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an M_r of 60,000. During further in vivo passaging, a virus that encodes an M_r-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.The independently isolated Friend and Rauscher erythroleukemia viruses (18, 48) are complexes of a replication competent murine leukemia virus (MuLV) and a replication-defective spleen focus-forming virus (SFFV) (39, 42, 47). The SFFVs encode Env glycoproteins (gp55) that are inefficiently processed to form larger cell surface derivatives (gp55^p) (19). The gp55 binds to erythropoietin receptors (EpoR) to cause erythroblast proliferation and splenomegaly in susceptible mice. Evidence has suggested that the critical mitogenic interaction occurs exclusively on cell surfaces (7, 16).SFFVs are structurally closely related to mink cell focus-inducing viruses (MCFs) (2, 5, 10, 50), a class of replication-competent murine retroviruses that has a broad host range termed polytropic (15, 21). Although MCFs are not inherited as replication-competent intact proviruses, the mouse genome contains numerous dispersed polytropic env gene sequences (8, 17, 27). MCFs apparently readily form de novo by recombination when ecotropic host range MuLVs replicate in mice (14, 15, 26, 43). MCF env genes typically are hybrid recombinants that contain a 5′ polytropic-specific region and a 3′ ecotropic-specific portion (26). They encode a gPr90 Env glycoprotein that is cleaved by partial proteolysis to form the products gp70 surface (SU) glycoprotein plus p15E transmembrane (TM) protein (32, 39, 47). In addition, MCFs often differ from ecotropic MuLVs in their long terminal repeat (LTR) sequences (8, 20, 26, 28, 29, 45).Based on their sequences, SFFVs could have derived from MCFs by a 585-base deletion and by a single-base addition in the ecotropic-specific portion of the env gene (10). Evidence suggests that both the 585-bp deletion and the frameshift mutation probably contribute to SFFV pathogenesis (3, 49). Several pathogenic differences among SFFV strains have also been ascribed to amino acid sequence differences in the ecotropic-specific portion of the Env glycoproteins (9, 41).This report describes the origin and rapid stepwise evolution of a new SFFV. This new pathogenic virus initially formed in a mouse that had been injected with an ecotropic strain of MuLV in the presence of a retroviral vector that does not encode any Env glycoprotein. The mouse developed erythroleukemia, splenomegaly, and polycythemia after a long lag phase. At that time the spleen contained viruses with env genes that were able to activate EpoR. Serial passage of this initial virus isolate resulted in selection of a novel SFFV that encodes a gp55 glycoprotein of 410 amino acids. This experimental system provides a method for isolating new SFFVs and for mapping the stages in their genesis.     

Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O₂ evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O₂ evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.Microalgae are able to efficiently convert sunlight, water, and CO₂ into a variety of products suitable for renewable energy applications, including H₂, carbohydrates, and lipids (11, 12, 16, 21, 38, 41, 44). The unicellular green alga Chlamydomonas reinhardtii has emerged as a model organism for studying algal physiology, photosynthesis, metabolism, nutrient stress, and the synthesis of bioenergy carriers (12, 15, 19, 24, 32). During acclimation to nitrogen deprivation, C. reinhardtii cells accumulate significant quantities of starch and form lipid bodies (4, 5, 8, 26, 28, 30, 34, 43, 46, 48). Despite the significance of these products in algal physiology and in biofuels applications, the metabolic, enzymatic, and regulatory mechanisms controlling the partitioning of metabolites into these distinct carbon stores in algae are poorly understood. Several C. reinhardtii starch mutants with various phenotypic changes in starch content and structure have been isolated (2,–4). Two of these, the sta6 and sta7 mutants, contain single-gene disruptions that result in “starchless” phenotypes with severely attenuated levels of starch granule accumulation (2, 4, 34, 39, 40, 48).The disrupted loci in the two isolated starchless mutants are distinct and each mutant has a unique phenotype (7, 40). In the sta6 mutant, the small, catalytic subunit of ADP-glucose pyrophosphorylase (AGPase-SS) is disrupted (2, 4, 48), and this mutant accumulates less than 1% of the starch observed in wild-type (WT) cells under conditions of nitrogen deprivation. The sta7 mutant contains a disrupted isoamylase gene (7, 8, 10, 39, 40) and also has severely attenuated levels of starch, but it accumulates a soluble glycogen-like product (4, 9). In this study, we conducted an examination of the unique physiological acclimations that are utilized by these mutants to adapt to the loss of starch synthesis. As the genetic lesions in these two mutants are distinct and block starch synthesis via two very different mechanisms, we investigated the physiological consequences of starch inhibition in both of these mutants from a holistic bioenergy perspective, which included photosynthetic parameters and the overall yields of lipids and carbohydrates, the two primary bioenergy carriers in C. reinhardtii. Specifically, we examined whether the inability to synthesize starch would result in the accumulation of additional lipid, alter cellular growth or cell size, affect acetate utilization, and/or influence photosynthetic O₂ evolution. Our data indicate that both the sta6 (BAFJ5) and sta7 (sta7-10) mutants accumulate more lipid than the CC124 control during nitrogen deprivation. However, the additional lipid does not completely offset the loss of starch synthesis from a complete energetic perspective. Increased lipid accumulation during nitrogen stress has also been reported for a variety of starch mutants in recent papers (26, 27, 46). A significant feature in both of the starchless mutants studied here is that O₂ evolution and acetate utilization are diminished during nitrogen stress, which is undesirable from an overall bioenergy perspective. Remarkably, complementation of sta7-10 with genomic DNA encoding the wild-type isoamylase gene resulted in cells that were larger than those of the sta6, sta7-10, and CC124 strains, exhibited the highest total lipid levels during nitrogen deprivation, and overaccumulated starch even in nutrient-replete medium.     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]