Evidence for the role of the light-harvesting chlorophyll protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II_α and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II_α centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II_α component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II_α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II_α and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II_α and PS II_β to the fluorescence induction kinetics. PS II_α characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

A cDNA clone encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley

Two cDNA clones for the barley photosystem I polypeptide which migrates with an apparent molecular mass of 9.5 kDa on SDS-polyacrylamide gels have been isolated using antibodies and an oligonucleotide probe. The determined N-terminal amino acid sequence for the mature polypeptide confirms the identification of the clones. The 644 base-pair sequence of one of the clones contains one large open reading frame coding for a 14 882 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 193 Da. The hydropathy plot of the polypeptide shows one membrane-spanning region with a predicted -helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Red algal LHC I genes have similarities with both Chl a/b- and a/c-binding proteins: A 21 kDa polypeptide encoded by LhcaR2 is one of the six LHC I polypeptides

Polypeptides from the PS I holocomplex of the red alga P. cruentum, purified for microsequencing, confirmed that six LHC I polypeptides from SDS-PAGE are distinct apoproteins. Analysis of a cDNA clone, designated as LhcaR2, from a cDNA library, indicates that it shares major structural features with the recently cloned first red algal gene LhcaR1. The LhcaR2 is believed to encode the 21.0 kDa polypeptide of the LHC I complex from comparison of the deduced amino acid sequence and the microsequences of several tryptic digest fragments from the isolated polypeptide. As in chlorophytic and chromophytic LHCs, the essential residues for Chl-binding and helix stabilization in helices 1 and 3 are highly conserved. Relatedness between rhodophytes and the chlorophytes is also inferred from sequence conservation in the N-flanking regions of helices 1 and 3. Conversely, helix 2 exhibited the highest similarity between LHC sequences of Chl a/c-binding chromophytes and the Chl a-binding rhodophytes, with 11 of 22 residues identical or conservatively substituted. Moreover, whereas in chlorophytes, the Q and E Chl-binding residues are separated by seven amino acid residues, they are always separated by 8 residues in rhodophytes and chromophytes. Superimposition of the predicted LhcaR2 sequence with the LHC II model [Kühlbrandt et al. (1994) Nature 367: 614–621] shows the same structural features except shortened connecting sequence between helices 1 and 2 on the lumenal side. The chimeric nature of rhodophyte genes, with both chromophytic and chlorophytic features, leads to the suggestion that they reflect attributes of an intermediate stage in LHC apoprotein evolution.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

The gene family coding for the light-harvesting polypeptides of Photosystem I of the red alga Galdieria sulphuraria*

Recently [Marquardt et al. (2000) Gene 255: 257–265], we isolated a gene encoding a polypeptide of the light-harvesting complex of Photosystem I (LHC I) of the red alga Galdieria sulphuraria. By screening a G. sulphuraria cDNA library with a DNA probe coding for the conserved first transmembrane helix of this protein we isolated four additional genes coding for LHC I polypeptides. The deduced preproteins had calculated molecular masses of 24.6–25.6 kDa and isoelectric points of 8.09–9.82. N-terminal sequencing of a LHC I polypeptide isolated by gel electrophoresis allowed us to determine the cleavage site of the transit peptide of one of the deduced polypeptides. The mature protein has a calculated molecular mass of 20.6 kDa and an isoelectric point of 7.76. The genes were amplified from nuclear G. sulphuraria DNA by polymerase chain reaction (PCR) using oligonucleotides annealing in the regions of the start and stop codons as primers. All genomic sequences were 80–300 base pairs longer than the PCR products obtained from the respective cDNA clones, pointing to the existence of 1–5 introns per gene. The G. sulphuraria genes form a homogeneous gene family with overall pairwise amino acid identities of 46.0–56.6%. Homology to two diatom, one cryptophytic and two higher plant light-harvesting polypeptides was lower with pairwise identities of 21.1–34.1%. Only one diatom polypeptide showed a higher degree of identity of up to −39.3%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Polypeptide composition,assembly and phosphorylation patterns of the photosystem II antenna system of Chlamydomonas reinhardtii

In recent years major progress has been made in describing the gene families that encode the polypeptides of the light-harvesting antenna system of photosystem II (PSII). At the same time, advances in the biochemical characterization of these antennae have been hampered by the high degree of similarity between the apoproteins. To help interpret the molecular results, we have re-examined the composition, the assembly and the phosphorylation patterns of the light-harvesting antenna of PSII (LHCII) in the green alga Chlamydomonas reinhardtii Dang, using a non-Tris SDS-PAGE system capable of resolving polypeptides that differ by as little as 200 daltons. Research to date has suggested that in C. reinhardtii the LHCII comprises just four polypeptides (p11, p13, p16 and p17), and CP29 and CP26 just one polypeptide each (p9 and p10, respectively), i.e. a total of six polypeptides. We report here that these antenna systems contain at least 15 polypeptides, 10 associated with LHCII, 3 with CP29, and 2 with CP26. All of these polypeptides have been positively identified by means of appropriate antibodies. We also demonstrate substantial heterogeneity to the pattern of in-vitro phosphorylation, with major differences found among members of closely spaced and immunologically related polypeptides. Most intriguing is the fact that the polypeptides that cross-react with the anti-type 2 LHCII antibodies of higher plants (p16, and to a lesser extent p11) are not phosphorylated, whereas in higher plants these are the most highly phosphorylated polypeptides. Also, unlike in higher plants, CP29 is heavily phosphorylated. Phosphorylation does not appear to have any effect on the mobility of polypeptides on fully denaturing SDS-PAGE gels. To learn more about the accumulation and organization of the light-harvesting polypeptides, we have also investigated a chlorophyll b-less mutant, cbn1-48. The LHCII is almost completely lost in this mutant, along with at least some LHCI. But the accumulation of CP29 and CP26 and their binding to PSII core complexes, is relatively unaffected. As expected, the loss of antenna polypeptides is accompanied by a reduction of the size of large reaction-center complexes. Following in-vitro phosphorylation the number of phosphorylated proteins is greatly increased in the mutant thylakoids compared to wildtype thylakoids. We present a model of the PSII antenna system to account for the new polypeptide complexity we have demonstrated.This work was supported by National Institute of Health grant GM22912 to L.A.S. We would like to thank Anastasios Melis for helpful discussions.     

Sequence conservation of light-harvesting and stress-response proteins in relation to the three-dimensional molecular structure of LHCII

The structure of pea light-harvesting complex LHCII determined to 3.4 Å resolution by electron crystallography (Kühlbrandt, Wang and Fujiyoshi (1994) Nature 367: 614–621) was examined to determine the relationship between structural elements and sequence motifs conserved in the extended family of light-harvesting antennas (Chl a/b, fucoxanthin Chl a/c proteins) and membrane-intrinsic stress-induced proteins (ELIPs) to which LHCII belongs. It is predicted that the eukaryotic ELIPs can bind at least four molecules of Chl. The one-helix prokaryotic ELIP of Synechococcus was modelled as a homodimer based on the high degree of conservation of residues involved in the interactions of the first (B) and third (A) helices of LHCII.Abbreviations CAB Chl a/b-binding - ELIP early light-inducible protein - FCP fucoxanthin-Chl a/c protein - Lut1, Lut2 lutein molecules 1 and 2     

Inhibition and promotion by light of the accumulation of translatable mRNA of the light-harvesting chlorophyll a/b-binding protein of photosystem II

The amount of in-vitro translatable mRNA of the light-harvesting chlorophyll a/b-binding protein (LHCP) of photosystem II strongly increases in darkness (D) after a 5-min red-light pulse while continuous illumination of mustard seedlings with far-red (FR), red or white light leads only to a slight increase in the amount of translatable LHCP-mRNA. No increase can be observed after a long-wavelength FR (RG9-light) pulse. However, a FR pretreatment prior to the RG9-light pulse strongly increase LHCP-mRNA accumulation in subsequent D. This is not observed in the case of the mRNA for the small subunit of ribulose-1.5-bisphosphate carboxylase. The increase of LHCP-mRNA in D after a FR pretreatment can be inhibited by a reillumination of the seedlings with FR. The inhibition of LHCP-mRNA accumulation during continuous illumination with FR and the strong increase in D following a FR illumination was found to be independent of chlorophyll biosynthesis since no correlation between chlorophyll biosynthesis and translatable LHCP-mRNA levels could be detected. Even strong changes in the amount of intermediates of chlorophyll biosynthesis caused by application of levulinic acid or 5-aminolevulinic acid did not affect LHCP-mRNA levels. Therefore, we conclude that the appearance of LHCP-mRNA is inhibited during continuous illumination, even though illumination leads to a storage of a light singal which promotes accumulation of translatable LHCP-mRNA in D.Abbreviations c continuous - Chl chlorophyll - D darkness - FR far-red light (3.5 W·m^-2) - LHCP light-harvesting chlorophyll a/b-binding protein of photosystem II - NF Norfluration - PChl protochlorophyll(ide) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W·m^-2) - RG9-light long-wavelength FR (10 W·m^-2) - SSU small subunit of ribulose-1.5-bisphosphate carboxylase - WL white light - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

A new member of the CAB gene family: structure,expression and chromosomal location of Cab-8, the tomato gene encoding the Type III chlorophyll a/b-binding polypeptide of photosystem I

We have previously reported the isolation and characterization of tomato nuclear genes encoding two types of chlorophyll a/b-binding (CAB) polypeptides localized in photosystem (PS) I and two types of CAB polypeptides localized in PSII. Sequence comparisons shows that all these genes are related to each other and thus belong to a single gene family. Here we report the isolation and characterization of an additional member of the tomato CAB gene family, the single tomato nuclear gene, designated Cab-8, which encodes a third type of CAB polypeptide localized in PSI. The protein encoded by Cab-8 is 65% and 60% divergent from the PSI Type I and Type II CAB polypeptides, respectively. The latter two are 65% divergent from each other. Only some short regions of the polypeptides are strongly conserved. The Cab-8 locus maps to chromosome 10, 9 map units from Cab-7, the gene encoding the Type II PSI CAB polypeptide. The Cab-8 gene contains two introns; the first intron matches in position the single intron in the Type II PSII CAB genes and the second intron matches in position the second intron in the Type II PSI CAB gene. Like other CAB genes, Cab-8 is light-regulated and is highly expressed in the leaf and to a lesser extent in other green organs.     

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII)

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.     

RT-PCR克隆籼稻叶绿素a/b结合蛋白基因全长cDNA及序列的in silico分析

根据日本晴cab4基因序列(GenBank:AK104499.1)设计引物,用RT-PCR的方法从籼稻9311中克隆了叶绿素a/b结合蛋白基因的全长cDNA,命名为cab-9311(cab gene from 9311)。insilico分析表明:cab-9311与cab4基因同源性为99%,编码的蛋白含有244个氨基酸,与cab4基因编码的蛋白同源性为98%。蛋白分子质量为26.9kD,理论等电点为6.52。第54位～第216位氨基酸是一个典型的叶绿素a/b结合蛋白功能域(chlorophyll a/bbinding domain)。跨膜分析和蛋白质三级预测显示,该蛋白在C端有一个典型的跨膜区。亚细胞定位分析表明该蛋白定位于叶绿体,是一个叶绿体内囊体膜上的锚定蛋白。     

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 °C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 °C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 °C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.     

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI) — Generation of protein sequence information

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.     

(Z)-11-Hexadecenyl compounds as attractants for male microlepidoptera of the subfamilies Argyresthiinae, Glyphipteryginae, and Crambinae

Traps baited with Z11-16:Ac, Z11-16:Ald, Z11-16:OH, and Z13-18:Ac, singly or in mixtures, were tested for their attractivity for males of microlepidoptera in an apple orchard, a mixed deciduous forest, and a coniferous forest. In particular, Z11-16:Ac and Z11-16: Ald attracted some Crambinae, one Glyphipteryx species, and several species of Argyresthia, including the injurous species A. fundella (on fir), A. conjugella (on apple), A. pruniella (on cherry), and A. thuiella (on Thuja).

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Attraction sexuelle de microlépidoptères des sous-familles Argyresthiinae, Glyphipteryginae et Crambinae par des hexadécènes 11Z

Résumé L'attraction de microlépidoptères par des hexadécènes 11Z a été examinée en 1982 et 1983 à l'aide de pièges sexuels placés dans différents biotopes de Hollande. Ces pièges contenaient différents mélanges synthétiques d'acétoxy-1-hexadécènes 11Z (Z11-16:Ac), d'hexadécènal 11Z (Z11-16:Ald), d'hexadécénol 11Z (Z11-16:OH) et d'acétoxyl-1-octadécène 13Z (Z13-18:Ac). Les pièges étaient placés dans un verger de pommiers, dans un bois de feuillus aux essences diverses et dans un bois de résineux. Des mâles de 10 espèces de lépidoptères ont été capturés avec ces mélanges, parmi lesquels ceux de 3 espèces de Pyralidae, d'une espèce du genre Glyphipteryx et de 6 espèces du genre Argyresthia. Parmi les Argyresthia, certaines espèces sont considérées comme des déprédateurs: A. fundella sur sapin (Abies), A. conjugella sur pommier (Malus), A. pruniella sur cerisier (Prunus) et A. thuiella sur Thuja. Enfin, un tableau fournit 28 produits contenant des hexadécènes Z11 actuellement connus comme attractifs sexuels de microlépidoptères; la majorité des espèces appartient aux sous-familles Crambinae, Argyresthiinae, Glyphipteryginae et Acrolepiinae.

     

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb)

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.