Nearest-neighbor analysis of higher-plant photosystem I holocomplex.

Photosystem 1 (PSI) preparations from barley (Hordeum vulgare) and spinach (Spinacia oleracea) were subjected to chemical cross-linking using the cleavable homobifunctional cross-linkers dithiobis(succinimidylpropionate) and 3,3'-dithiobis(sulfosuccinimidyl-propionate). The overall pattern of cross-linked products was analyzed by the simple but powerful technique of diagonal electrophoresis, in which the disulfide bond in the cross-linker was cleaved between the first and second dimensions of the gel, and immunoblotting. A large number of cross-linked products were identified. Together with preexisting data on the structure of PSI, it was deduced that the subunits PSI-D, PSI-H, PSI-I, and PSI-L occupy one side of the complex, whereas PSI-E, PSI-F, and PSI-J occupy the other. PSI-K and PSI-G appear to be adjacent to Lhca3 and Lhca2, respectively, and not close to the other small subunits. Experiments with isolated light-harvesting complex I preparations indicate that the subunits are organized as dimers, which seem to associate to the PSI-A/PSI-B proteins independent of each other. We suggest which PSI subunit corresponds to each membrane-spanning helix in the cyanobacterial PSI structure, and present a model for higher-plant PSI.     

Structural and functional analysis of the reducing side of photosystem I

Structural analysis of the reducing side of photosystem I (PSI) has been carried out using chemical cross-linking and monospecific antibodies. Incubation of PSI isolated from barley (Hordeum vulgare L.) with the hydrophilic cross-linking agent N-ethyl-3-[3-(dimethylamino) propyl]-carbodiimide leads to cross-linking of the PSI-D subunit with the PSI-E and PSI-H subunits. In the presence of ferredoxin, cross-linking results in the formation of cross-linked products composed of PSI-D, PSI-E and ferredoxin and in a block in steady state NADP⁺ photoreduction. No cross-linking of ferredoxin occurs at elevated ionic strength or using heat-denatured ferredoxin. Cross-linking of ferredoxin does not inhibit electron transfer from plastocyanin to methyl viologen. Steady state NADP⁺ photoreduction was analyzed in PSI or thyla-koids incubated with antibodies against individual PSI subunits. Incubation with antibodies against PSI-C, -H, -I, or -L had no effect on PSI activity, whereas antibodies against PSI-D or PSI-E had similar effects and caused a large decrease in activity. The results provide evidence that the PSI-D and PSI-E subunits are localized on the reducing side of PSI, forming a barrier between PSI-C and the stroma as well as a docking site for ferredoxin. The PSI-H subunit has an exposed, stromal domain but this does not appear to contribute to the ferredoxin docking.     

Reconstitution of barley photosystem I reveals that the N-terminus of the PSI-D subunit is essential for tight binding of PSI-C

Removal of the peripheral subunits PSI-C, -D and -E from the photosystem I (PSI) complex of barley requires a urea treatment much harsher than required to remove the similar subunits from cyanobacterial PSI. The resulting PSI barley core was reconstituted by addition of the E. coli expressed subunits PSI-C and -D, and PSI-E isolated from barley. Western blotting, flash photolysis and NADP⁺ photoreduction measurements demonstrated complete and specific removal of the three subunits from the core and efficient reconstitution of the complex after addition of PSI-C, -D and -E. Flash photolysis reveals that PSI-D is essential for binding of functional PSI-C to the PSI core. An N-terminally truncated barley PSI-D lacking 24 amino acid residues and thus being without the N-terminal extension characteristic for higher plant PSI-D proteins reconstitutes the PSI core to 50% of the level obtained with intact PSI-D as demonstrated by flash photolysis and NADP⁺ photoreduction measurements. Cyanobacterial PSI-D is functionally equivalent to truncated barley PSI-D with respect to its activity to reconstitute the PSI core. This shows that the N-terminal extension of plant PSI-D plays a key role in binding PSI-C to the core. The plant-specific N-terminus of PSI-D is hypothesized to execute its function through interaction with a plant-specific PSI subunit, possibly PSI-H. An anchoring function of the N-terminus of PSI-D would also explain the harsh treatment needed to obtain a plant PSI core. PSI-E is important for efficient NADP⁺ reduction but does not influence electron transfer to iron-sulphur centres A/B nor binding of PSI-C. The enhancing effect of PSI-E on NADP⁺ reduction is independent of the presence of the N-terminus of PSI-D.     

Molecular aspects of photosystem I

Photosystem I (PSI) in higher plants consists of 17 polypeptide subunits. Cofactors are chlorophyll a and b , β-carotene, phylloquinone and iron-sulfur clusters. Eight subunits are specific for higher plants while the remaining ones are also present in cyanobacteria. Two 80-kDa subunits (PSI-A and -B) constitute the major part of PSI and bind most of the pigments and electron donors and acceptors. The 9-kDa PSI-C carries the remaining electron acceptors which are [4Fe-4S] iron sulfur clusters. PSI-D, -E and -H have importance for integrity and function at the stromal face of PSI while PSI-F has importance for function at the lumenal face. PSI-N is localized at the lumenal side, but its function is unknown. Four subunits are light-harvesting chlorophyll a/b -binding proteins. The remaining subunits are integral membrane proteins with poorly understood function. Subunit interactions have been studied in reconstitution experiments and by cross-linking studies. Based on these data, it is concluded that iron-sulfur cluster F_B is proximal to F_X and that F_A is the terminal acceptor in PSI. Similarities between PSI and the reaction center from green sulfur bacteria are discussed.     

Photoinhibition of Photosystem I damages both reaction centre proteins PSI-A and PSI-B and acceptor-side located small Photosystem I polypeptides

Photoinhibition of Photosystem I at chilling temperatures was investigated. Illumination of barley and cucumber leaves at 4°C induced a lowered Photosystem I activity. In barley, the reaction centre proteins PSI-A and PSI-B were both partially degraded as was the nuclear-encoded PSI-D polypeptide. Barley leaves infiltrated with KCN to increase oxidative stress, showed increased photoinhibition of Photosystem I, including reduced photochemical activity and marked degradation of several Photosystem I polypeptides. The most rapid and pronounced degradation was found in the PSI-D and PSI-E polypeptides exposed at the Photosystem I acceptor side. The PSI-A, -B, -C, -G, -H, -K and -L polypeptides were less extensively damaged. No damage of the lumenally oriented PSI-F and -N polypeptides was detected. The elevated photoinhibition of Photosystem I seen in KCN treated barley is most likely induced by a combination of increased active oxygen due to inhibited scavenging and increased accumulation of reducing power due to inhibition of the Calvin cycle. In barley, photo-inactivation of Photosystem I closely followed the degradation of PSI-A and PSI-B. Illumination of cucumber resulted in a pronounced loss of activity and appearance of specific PSI-A and PSI-B degradation products whereas the total PSI-A/B degradation was small. The PSI-A/B degradation identified in barley is interpreted to reflect a physiologically relevant process being part of a repair cycle, whereas the much smaller PSI-A/B degradation observed in cucumber is interpreted to represent an irreversible damage induced far below the temperature tolerance for cucumber.     

Arabidopsis thaliana plants lacking the PSI-D subunit of photosystem I suffer severe photoinhibition,have unstable photosystem I complexes,and altered redox homeostasis in the chloroplast stroma

The PSI-D subunit of photosystem I is a hydrophilic subunit of about 18 kDa, which is exposed to the stroma and has an important function in the docking of ferredoxin to photosystem I. We have used an antisense approach to obtain Arabidopsis thaliana plants with only 5-60% of PSI-D. No plants were recovered completely lacking PSI-D, suggesting that PSI-D is essential for a functional PSI in plants. Plants with reduced amounts of PSI-D showed a similar decrease in all other subunits of PSI including the light harvesting complex, suggesting that in the absence of PSI-D, PSI cannot be properly assembled and becomes degraded. Plants with reduced amounts of PSI-D became light-stressed even in low light although they exhibited high non-photochemical quenching (NPQ). The high NPQ was generated by upregulating the level of violaxanthin de-epoxidase and PsbS, which are both essential components of NPQ. Interestingly, the lack of PSI-D affected the redox state of thioredoxin. During the normal light cycle thioredoxin became increasingly oxidized, which was observed as decreasing malate dehydrogenase activity over a 4-h light period. This result shows that photosynthesis was close to normal the first 15 min, but after 2-4 h photoinhibition dominated as the stroma progressively became less reduced. The change in the thiol disulfide redox state might be fatal for the PSI-D-less plants, because reduction of thioredoxin is one of the main switches for the initiation of CO2 assimilation and photoprotection upon light exposure.     

Mutants for photosystem I subunit D of Arabidopsis thaliana: effects on photosynthesis, photosystem I stability and expression of nuclear genes for chloroplast functions

In Arabidopsis thaliana, the D-subunit of photosystem I (PSI-D) is encoded by two functional genes, PsaD1 and PsaD2, which are highly homologous. Knock-out alleles for each of the loci have been identified by a combination of forward and reverse genetics. The double mutant psad1-1 psad2-1 is seedling-lethal, high-chlorophyll-fluorescent and deficient for all tested PSI subunits, indicating that PSI-D is essential for photosynthesis. In addition, psad1-1 psad2-1 plants show a defect in the accumulation of thylakoid multiprotein complexes other than PSI. Of the single-gene mutations, psad2 plants behave like wild-type (WT) plants, whereas psad1-1 markedly affects the accumulation of PsaD mRNA and protein, and photosynthetic electron flow. Additional effects of the psad1-1 mutation include a decrease in growth rate under greenhouse conditions and downregulation of the mRNA expression of most genes involved in the light phase of photosynthesis. In the same mutant, a marked decrease in the levels of PSI and PSII polypeptides is evident, as well as a light-green leaf coloration and increased photosensitivity. Increased dosage of PsaD2 in the psad1-1 background restores the WT phenotype, indicating that PSI-D1 and PSI-D2 have redundant functions.     

In vitro proteolytic processing of pea and jack bean storage proteins by an endopeptidase from lupin seeds

The highly specific proteolytic breakdown observed upon prolonged treatment of pea legumin and pea and jack bean vicilin with a thiol endopeptidase purified from mature lupin seeds has been studied in detail. Proteolytic cleavage occurred in the acidic subunits of pea legumin, whereas the basic subunits were unaffected. Jack bean vicilin (M, 47 K) was cleaved near the middle of the polypeptide chain, whereas pea vicilin (M, 50 K) was cleaved into two fragments of M, 30 K and 20 K, respectively. The 30 K M, polypeptide chain contained covalently linked carbohydrate and had an N-terminal sequence suggesting that cleavage had taken place between the α and β region of the vicilin 50 K M, polypeptide as previously described in vivo. These results suggested that the cleavage specificity of lupin endopeptidase was in the proximity of paired arginine amino acid residues.The changes in the vicilin polypeptides due to proteolytic cleavage by lupin enzyme and those occurring during germination of pea seeds are also reported and discussed.     

Isolation and characterization of PSI–LHCI super-complex and their sub-complexes from a red alga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>

Photosystem I (PSI)–light-harvesting complex I (LHCI) super-complex and its sub-complexes PSI core and LHCI, were purified from a unicellular red alga Cyanidioschyzon merolae and characterized. PSI–LHCI of C. merolae existed as a monomer with a molecular mass of 580 kDa. Mass spectrometry analysis identified 11 subunits (PsaA, B, C, D, E, F, I, J, K, L, O) in the core complex and three LHCI subunits, CMQ142C, CMN234C, and CMN235C in LHCI, indicating that at least three Lhcr subunits associate with the red algal PSI core. PsaG was not found in the red algae PSI–LHCI, and we suggest that the position corresponding to Lhca1 in higher plant PSI–LHCI is empty in the red algal PSI–LHCI. The PSI–LHCI complex was separated into two bands on native PAGE, suggesting that two different complexes may be present with slightly different protein compositions probably with respective to the numbers of Lhcr subunits. Based on the results obtained, a structural model was proposed for the red algal PSI–LHCI. Furthermore, pigment analysis revealed that the C. merolae PSI–LHCI contained a large amount of zeaxanthin, which is mainly associated with the LHCI complex whereas little zeaxanthin was found in the PSI core. This indicates a unique feature of the carotenoid composition of the Lhcr proteins and may suggest an important role of Zea in the light-harvesting and photoprotection of the red algal PSI–LHCI complex.     

Identification of surface-exposed domains on the reducing side of photosystem I.

Photosystem I (PSI) is a multisubunit enzyme that catalyzes the light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobacterium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low concentration of endoproteinase glutamic acid (Glu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. Glu63 and Glu67, located near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cleaved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Glu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, several secondary cleavage sites on PsaD, PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD and PsaE were intact. Glu88 and Glu124 of PsaF became susceptible to endoproteinase Glu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase Glu-C or thermolysin showed that the intact PsaE and PsaD, but not their major degradation products lacking C-terminal domains, were heavily biotinylated. Therefore, lysine-74 at the C terminus of PsaE was accessible for biotinylation. Similarly, lysine-107, or lysine-118, or both in PsaD could be modified by NHS-biotin.     

Susceptibility to endoglycosidase F and H at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits

We have studied the differential susceptibility to endoglycosidase F and H of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]mannose for 6 h. [3H]Man-labeled TSH and free alpha-subunits were obtained from homogenates using specific antisera and were digested with endoglycosidase F and H in their native states or after heat-denaturation and reduction in the presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that effects of endoglycosidase at the individual glycosylation sites could be determined. There was very little preferential cleavage by endoglycosidase H and F among the glycosylation sites of TSH subunits. Endoglycosidase F treatment of native free alpha-subunits showed slight preferential cleavage at Asn 82 of alpha-subunits after a 4 h incubation, whereas endoglycosidase H cleaved oligosaccharides equally well at Asn 56 and Asn 82. The Asn 82 oligosaccharide of native TSH heterodimers was also slightly preferentially cleaved by endoglycosidase F, but endoglycosidase H cleaved oligosaccharides equally well at all TSH glycosylation sites. Heat denaturation, reduction and the presence of detergent did not alter this slight preferential cleavage by endoglycosidase F at Asn 82 of alpha-subunits, suggesting that the primary structures of the TSH subunits in part influenced the efficiency of enzyme action at specific sites. Thus, the susceptibility to endoglycosidase F differs very slightly at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these small differences could be due to properties of either the enzyme or substrates.     

Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirus and enterovirus IRES-driven translation and cleave several proteins of cellular and viral origin.

Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognate internal ribosome entry segment (IRES) (S. J. Hambidge and P. Sarnow, Proc. Natl. Acad. Sci. USA 89:10272-10276, 1992; H.-D. Liebig, E. Ziegler, R. Yan, K. Hartmuth, H. Klump, H. Kowalski, D. Blaas, W. Sommergruber, L. Frasel, B. Lamphear, R. Rhoads, E. Kuechler, and T. Skern, Biochemistry 32:7581-7588, 1993). Given the functional similarities between the foot-and-mouth disease virus (FMDV) L proteinase and these 2A proteinases (autocatalytic excision from the nascent viral polyprotein and cleavage of eIF-4 gamma), we investigated whether the FMDV L proteinase would also be able to stimulate translation initiation. We found that purified recombinant FMDV Lb proteinase could stimulate in vitro translation driven from a rhinovirus or enterovirus IRES by 5- to 10-fold. In contrast, stimulation of translation initiation on a cardiovirus IRES by this proteinase was minimal, and stimulation of translation driven from the cognate FMDV IRES could not be evidenced. Studies using an inhibitor or a mutant Lb proteinase indicated that stimulation of IRES-driven translation is mediated via proteolysis of some cellular component(s). Our studies also demonstrated that the Lb proteinase is capable of stimulating initiation of translation on an uncapped cellular message. Unexpectedly, and in contrast to the 2A proteinases, the Lb proteinase specifically cleaved the products of the two reporter genes used in this study: Xenopus laevis cyclin B2 and influenza virus NS. Therefore, we also set out to investigate the requirements for substrate recognition by the Lb proteinase. Purified recombinant Lb proteinase recognized at least one mengovirus polypeptide and specifically cleaved human cyclin A and poliovirus replicase-related polypeptides. In the latter case, the site(s) of cleavage was located within the N-terminal part of polypeptide 3D. Sequence comparisons revealed no significant primary sequence similarities between the target proteins and the two sites already known to be recognized by the FMDV L proteinase.     

Heat-induced disassembly and degradation of chlorophyll-containing protein complexes in vivo

Gradual heating of green leaves up to non-physiological temperatures is often used to estimate thermal stability of photosynthetic apparatus. However, a complete sequence of heat-induced disassembly and denaturation of chlorophyll-containing protein complexes (CPCs) has not been reported yet. In this work, we heated (1 °C·min^− 1) barley leaves to temperatures selected according to the changes in the chlorophyll fluorescence temperature curve (FTC) and we analyzed CPC stability by two-dimensional native Deriphat/SDS-PAGE. The first distinct change in both structure and function of photosystem II (PSII) appeared at 40-50 °C. PSII core (CCII) dimers began to dissociate monomers, which was accompanied by a decrease in PSII photochemistry and reflected in FTC as the first fluorescence increase. Further changes in CPCs appeared at 57-60 °C, when FTC increases to its second maximum. Photosystem I (PSI) cores (CCI) partially dissociated from light-harvesting complexes of PSI (LHCI) and formed aggregates. The rest of CCI-LHCI complexes, as well as the CCI aggregates, degraded to the PSI-A/B heterodimer in leaves heated to 70 °C. Heating to these temperatures led to a complete degradation of CCII components and corresponding loss of PSII photochemistry. Trimeric light-harvesting complexes of PSII (LHCII) markedly dissociated to monomers and denatured, as evidenced by a release of large amount of free chlorophylls. Between 70 and 80 °C, a complete degradation of LHCII occurred, leaving the PSI-A/B heterodimer as the only detectable CPC in the membrane. This most thermostable CPC disappeared after heating to 90 °C, which corresponded to a loss of PSI photochemistry.     

Single and double knockouts of the genes for photosystem I subunits G,K, and H of Arabidopsis. Effects on photosystem I composition,photosynthetic electron flow,and state transitions

Photosystem I (PSI) of higher plants contains 18 subunits. Using Arabidopsis En insertion lines, we have isolated knockout alleles of the genes psaG, psaH2, and psaK, which code for PSI-G, -H, and -K. In the mutants psak-1 and psag-1.4, complete loss of PSI-K and -G, respectively, was confirmed, whereas the residual H level in psah2-1.4 is due to a second gene encoding PSI-H, psaH1. Double mutants, lacking PSI-G, and also -K, or a fraction of -H, together with the three single mutants were characterized for their growth phenotypes and PSI polypeptide composition. In general, the loss of each subunit has secondary, in some cases additive, effects on the abundance of other PSI polypeptides, such as D, E, H, L, N, and the light-harvesting complex I proteins Lhca2 and 3. In the G-less mutant psag-1.4, the variation in PSI composition suggests that PSI-G stabilizes the PSI-core. Levels of light-harvesting complex I proteins in plants, which lack simultaneously PSI-G and -K, indicate that PSI subunits other than G and K can also bind Lhca2 and 3. In the same single and double mutants, psag-1.4, psak-1, psah2-1.4, psag-1.4/psah2-1.4, and psag-1.4/psak-1 photosynthetic electron flow and excitation energy quenching were analyzed to address the roles of the various subunits in P700 reduction (mediated by PSI-F and -N) and oxidation (PSI-E), and state transitions (PSI-H). Based on the results, we also suggest for PSI-K a role in state transitions.     

Subunit arrangement in V-ATPase from Thermus thermophilus

The V0V1-ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V1 part (ABDF) and a proton channel V0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V0V1-ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI. Exposure of the isolated V0 to acid or 8 m urea also produced two subcomplexes, EGI and CL. Thus, the C subunit (homologue of d subunit, yeast Vma6p) associates with the L subunit ring tightly, and I (homologue of 100-kDa subunit, yeast Vph1p), E, and G subunits constitute a stable complex. Based on these observations and our recent demonstration that D, F, and L subunits rotate relative to A3B3 (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315; Yokoyama, K., Nakano, M., Imamura, H., Yoshida, M., and Tamakoshi, M. (2003) J. Biol. Chem. 278, 24255-24258), we propose that C, D, F, and L subunits constitute the central rotor shaft and A, B, E, G, and I subunits comprise the surrounding stator apparatus in the V0V1-ATPase.     

Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana

Chloroplasts are central to the provision of energy for green plants. Their photosynthetic membrane consists of two major complexes converting sunlight: photosystem I (PSI) and photosystem II (PSII). The energy flow toward both photosystems is regulated by light-harvesting complex II (LHCII), which after phosphorylation can move from PSII to PSI in the so-called state 1 to state 2 transition and can move back to PSII after dephosphorylation. To investigate the changes of PSI and PSII during state transitions, we studied the structures and frequencies of all major membrane complexes from Arabidopsis thaliana chloroplasts at conditions favoring either state 1 or state 2. We solubilized thylakoid membranes with digitonin and analyzed the complete set of complexes immediately after solubilization by electron microscopy and image analysis. Classification indicated the presence of a PSI-LHCII supercomplex consisting of one PSI-LHCI complex and one LHCII trimer, which was more abundant in state 2 conditions. The presence of LHCII was confirmed by excitation spectra of the PSI emission of membranes in state 1 or state 2. The PSI-LHCII complex could be averaged with a resolution of 16 A, showing that LHCII has a specific binding site at the PSI-A, -H, -L, and -K subunits.     

Antiapoptotic herpesvirus Bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins

The antiapoptotic Bcl-2 and Bcl-x(L) proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (E. H.-Y. Cheng, D. G. Kirsch, R. J. Clem, R. Ravi, M. B. Kastan, A. Bedi, K. Ueno, and J. M. Hardwick, Science 278:1966-1968, 1997; R. J. Clem, E. H.-Y. Cheng, C. L. Karp, D. G. Kirsch, K. Ueno, A. Takahashi, M. B. Kastan, D. E. Griffin, W. C. Earnshaw, M. A. Veliuona, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 95:554-559, 1998). Gamma herpesviruses also encode homologs of the Bcl-2 family. All tested herpesvirus Bcl-2 homologs possess antiapoptotic activity, including the more distantly related homologs encoded by murine gammaherpesvirus 68 (gammaHV68) and bovine herpesvirus 4 (BHV4), as described here. To determine if viral Bcl-2 proteins can be converted into death factors, similar to their cellular counterparts, five herpesvirus Bcl-2 homologs from five different viruses were tested for their susceptibility to caspases. Only the viral Bcl-2 protein encoded by gammaHV68 was susceptible to caspase digestion. However, unlike the caspase cleavage products of cellular Bcl-2, Bcl-x(L), and Bid, which are potent inducers of apoptosis, the cleavage product of gammaHV68 Bcl-2 lacked proapoptotic activity. KSBcl-2, encoded by the Kaposi's sarcoma-associated herpesvirus, was the only viral Bcl-2 homolog that was capable of killing cells when expressed as an N-terminal truncation. However, because KSBcl-2 was not cleavable by caspases, the latent proapoptotic activity of KSBcl-2 apparently cannot be released. The Bcl-2 homologs encoded by herpesvirus saimiri, Epstein-Barr virus, and BHV4 were not cleaved by apoptotic cell extracts and did not possess latent proapoptotic activities. Thus, herpesvirus Bcl-2 homologs escape negative regulation by retaining their antiapoptotic activities and/or failing to be converted into proapoptotic proteins by caspases during programmed cell death.     

Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin

Limited treatment of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of the Mr 98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates of Mr 60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as where T1 and T2 indicate tryptic cleavage sites; cleavage at only T1 or T2 gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping using Staphylococcus aureus V8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (approximately 20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on the Km for Glc or the Ki for Glc-6-P, though a slight decrease in Km for ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.     

Identification and analysis of the maize P700 chlorophyll a apoproteins PSI-A1 and PSI-A2 by high pressure liquid chromatography analysis and partial sequence determination

We recently described a pair of partially homologous maize chloroplast genes, one of which was shown to code for an apoprotein of the P700 chlorophyll a complex of photosystem I (Fish, L.E., Kück, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). Two chlorophyll-free apoprotein bands from maize chlorophyll-protein complex I (CPI) can be resolved on lithium dodecyl sulfate (LDS)-urea polyacrylamide gels. Proteins in both bands react with antibodies prepared against CPI, but antibodies prepared against two synthetic peptides corresponding to predicted sequences of PSI-A1 react only with the upper band. The presence of products of the two genes, ps1A1 and ps1A2, in CPI was verified by analysis of cyanogen bromide (CNBr) fragments of the lower apoprotein band obtained from LDS-urea polyacrylamide gels by reverse-phase high pressure liquid chromatography. Amino-terminal sequencing of five CNBr fragments indicates that the lower band contains a product of the ps1A2 gene. The possibility of extensive processing was investigated because the apparent molecular masses of the maize CPI proteins are about 58-70 kDa on LDS-polyacrylamide gels rather than the predicted sizes of about 83 kDa. Antibodies against a synthetic peptide corresponding to a predicted sequence in PSI-A1 were used to determine that the amino-terminal end of PSI-A1 is intact beyond about position 52. The amino-terminal CNBr fragment of PSI-A2 was identified by sequencing, indicating that the amino-terminal end of PSI-A2 is not processed. The carboxyl-terminal CNBr fragment of PSI-A2 was also identified by sequencing. These results indicate that the PSI-A1 and PSI-A2 polypeptides are not extensively processed, although some processing at the carboxyl-terminal end has not been ruled out.     

Competition between linear and cyclic electron flow in plants deficient in Photosystem I

Photosynthetic electron transport can involve either a linear flow from water to NADP, via Photosystems (PS) II and I or a cyclic flow just involving PSI. Little is known about factors regulating the relative flow through each of these pathways. We have examined photosynthetic electron transport through each system in plants of Arabidopsis thaliana in which either the PSI-D1 or PSI-E1 subunits of PSI have been knocked out. In both cases, this results in an imbalance in the turnover of PSI and PSII, such that PSII electron transport is limited by PSI turnover. Phosphorylation of light-harvesting complex II (LHCII) and its migration to PSI is enhanced but only partially reversible and not sufficient to balance photosystem turnover. In spite of this, cyclic electron flow is able to compete efficiently with PSI across a range of conditions. In dark-adapted leaves, the efficiency of cyclic relative to linear flow induced by far-red light is increased, implying that the limiting step of cyclic flow lies in the re-injection of electrons into the electron transport chain. Illumination of leaves with white light resulted in transient induction of a significant non-photochemical quenching in knockout plants which is probably high energy state quenching induced by cyclic electron flow. At high light and at low CO₂, non-photochemical quenching was greater in the knockout plants than in the wildtype. Comparison of PSI and PSII turnover under such conditions suggested that this is generated by cyclic electron flow around PSI. We conclude that, when the concentration of PSI is limiting, cyclic electron flow is still able to compete effectively with linear flow to maintain a high ΔpH to regulate photosynthesis.