Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat1

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.

Implications of a Developmental-Stage-Dependent Thylakoid-Bound Protease in the Stabilization of the Light-Harvesting Pigment-Protein Complex Serving Photosystem II during Thylakoid Biogenesis in Red Kidney Bean1

Intact etioplasts of bean (Phaseolus vulgaris) plants exhibit proteolytic activity against the exogenously added apoprotein of the light-harvesting pigment-protein complex serving photosystem II (LHCII) that increases as etiolation is prolonged. The activity increases in the membrane fraction but not in the stroma, where it remains low and constant and is mainly directed against LHCII and protochlorophyllide oxidoreductase. The thylakoid proteolytic activity, which is low in etioplasts of 6-d-old etiolated plants, increases in plants pretreated with a pulse of light or exposed to intermittent-light (ImL) cycles, but decreases during prolonged exposure to continuous light, coincident with chlorophyll (Chl) accumulation. To distinguish between the control of Chl and/or development on proteolytic activity, we used plants exposed to ImL cycles of varying dark-phase durations. In ImL plants exposed to an equal number of ImL cycles with short or long dark intervals (i.e. equal Chl accumulation but different developmental stage) proteolytic activity increased with the duration of the dark phase. In plants exposed to ImL for equal durations to such light-dark cycles (i.e. different Chl accumulation but same developmental stage) the proteolytic activity was similar. These results suggest that the protease, which is free to act under limited Chl accumulation, is dependent on the developmental stage of the chloroplast, and give a clue as to why plants in ImL with short dark intervals contain LHCII, whereas those with long dark intervals possess only photosystem-unit cores and lack LHCII.

Biochemical Characterization of Stromal and Thylakoid-Bound Isoforms of Isoprene Synthase in Willow Leaves1

Isoprene synthase is the enzyme responsible for the foliar emission of the hydrocarbon isoprene (2-methyl-1,3-butadiene) from many C3 plants. Previously, thylakoid-bound and soluble forms of isoprene synthase had been isolated separately, each from different plant species using different procedures. Here we describe the isolation of thylakoid-bound and soluble isoprene synthases from a single willow (Salix discolor L.) leaf-fractionation protocol. Willow leaf isoprene synthase appears to be plastidic, with whole-leaf and intact chloroplast fractionations yielding approximately equal soluble (i.e. stromal) and thylakoid-bound isoprene synthase activities. Although thylakoid-bound isoprene synthase is tightly bound to the thylakoid membrane (M.C. Wildermuth, R. Fall [1996] Plant Physiol 112: 171–182), it can be solubilized by pH 10.0 treatment. The solubilized thylakoid-bound and stromal isoprene synthases exhibit similar catalytic properties, and contain essential cysteine, histidine, and arginine residues, as do other isoprenoid synthases. In addition, two regulators of foliar isoprene emission, leaf age and light, do not alter the percentage of isoprene synthase activity in the bound or soluble form. The relationship between the isoprene synthase isoforms and the implications for function and regulation of isoprene production are discussed.

ATP bound to the origin recognition complex is important for preRC formation

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

Signal termination in bacterial chemotaxis: CheZ mediates dephosphorylation of free rather than switch-bound CheY.

Chemotaxis in bacteria is controlled by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein CheY. When phosphorylated, CheY binds to FliM, which is one of the proteins that constitute the "gear box" (or "switch") of the flagellar motor. Consequently, the motor shifts from the default direction of rotation, counterclockwise, to clockwise rotation. This biased rotation is terminated when CheY is dephosphorylated either spontaneously or, faster, by a specific phosphatase, CheZ. Logically, one might expect CheZ to act directly on FliM-bound CheY. However, here we provide direct biochemical evidence that, in contrast to this expectation, phosphorylated CheY (CheY approximately P), bound to FliM, is protected from dephosphorylation by CheZ. The complex between CheY approximately P and FliM was trapped by cross-linking with dimethylsuberimidate, and its susceptibility to CheZ was measured. CheY approximately P complexed with FliM, unlike free CheY approximately P, was not dephosphorylated by CheZ. However, it did undergo spontaneous dephosphorylation. Nonspecific cross-linked CheY dimers, measured as a control, were dephosphorylated by CheZ. No significant binding between CheZ and any of the switch proteins was detected. It is concluded that, in the termination mechanism of signal transduction in bacterial chemotaxis, CheZ acts only on free CheY approximately P. We suggest that CheZ affects switch-bound CheY approximately P by shifting the equilibrium between bound and free CheY approximately P.

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Substrate-bound agrin induces expression of acetylcholine receptor epsilon-subunit gene in cultured mammalian muscle cells.

Expression of the epsilon-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with epsilon-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of epsilon-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina.

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

Reaction of the Escherichia coli quinol oxidase cytochrome bo3 with dioxygen: the role of a bound ubiquinone molecule.

We have studied the kinetics of the oxygen reaction of the fully reduced quinol oxidase, cytochrome bo3, using flow-flash and stopped flow techniques. This enzyme belongs to the heme-copper oxidase family but lacks the CuA center of the cytochrome c oxidases. Depending on the isolation procedure, the kinetics are found to be either nearly monophasic and very different from those of cytochrome c oxidase or multiphasic and quite similar to cytochrome c oxidase. The multiphasic kinetics in cytochrome c oxidase can largely be attributed to the presence Of CuA as the donor of a fourth electron, which rereduces the originally oxidized low-spin heme and completes the reduction of O2 to water. Monophasic kinetics would thus be expected, a priori, for cytochrome bo3 since it lacks the CuA center, and in this case we show that the oxygen reaction is incomplete and ends with the ferryl intermediate. Multiphasic kinetics thus suggest the presence of an extra electron donor (analogous to CuA). We observe such kinetics exclusively with cytochrome bo3 that contains a single equivalent of bound ubiquinone-8, whereas we find no bound ubiquinone in an enzyme exhibiting monophasic kinetics. Reconstitution with ubiquinone-8 converts the reaction kinetics from monophasic to multiphasic. We conclude that a single bound ubiquinone molecule in cytochrome bo3 is capable of fast rereduction of heme b and that the reaction with O2 is quite similar in quinol and cytochrome c oxidases.

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.