Interhelical interactions between D92 and C218 in the cytoplasmic domain regulate proton uptake upon N-decay in the proton transport of Acetabularia rhodopsin II.

Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.

Hybrid Indicators for Fast and Sensitive Voltage Imaging.

Membrane voltage is an important biophysical signal that underlies intercellular electrical communications. A fluorescent voltage indicator is presented that enables the investigation of electrical signaling at high spatial resolution. The method is built upon the site-specific modification of microbial rhodopsin proteins with organic fluorophores, resulting in a hybrid indicator scaffold that is one of the most sensitive and fastest orange-colored voltage indicators developed to date. We applied this technique to optically map electrical connectivity in cultured cells, which revealed gap junction-mediated long-range coupling that spanned over hundreds of micrometers.

Interaction with plant transcription factors can mediate nuclear import of phytochrome B.

Phytochromes (phy) are red/far-red-absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N- and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.

Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum.

Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.

Crystal structure of the eukaryotic light-driven proton-pumping rhodopsin, Acetabularia rhodopsin II, from marine alga.

Acetabularia rhodopsin (AR) is a rhodopsin from the marine plant Acetabularia acetabulum. The opsin-encoding gene from A. acetabulum, ARII, was cloned and found to be novel but homologous to that reported previously. ARII is a light-driven proton pump, as demonstrated by the existence of a photo-induced current through Xenopus oocytes expressing ARII. The photochemical reaction of ARII prepared by cell-free protein synthesis was similar to that of bacteriorhodopsin (BR), except for the lack of light-dark adaptation and the different proton release and uptake sequence. The crystal structure determined at 3.2 Å resolution is the first structure of a eukaryotic member of the microbial rhodopsin family. The structure of ARII is similar to that of BR. From the cytoplasmic side to the extracellular side of the proton transfer pathway in ARII, Asp92, a Schiff base, Asp207, Asp81, Arg78, Glu199, and Ser189 are arranged in positions similar to those of the corresponding residues directly involved in proton transfer by BR. The side-chain carboxyl group of Asp92 appears to interact with the sulfhydryl group of Cys218, which is unique to ARII and corresponds to Leu223 of BR and to Asp217 of Anabaena sensory rhodopsin. The orientation of the Arg78 side chain is opposite to the corresponding Arg82 of BR. The putative absence of water molecules around Glu199 and Arg78 may disrupt the formation of the low-barrier hydrogen bond at Glu199, resulting in the "late proton release".

Dynamics of voltage profile in enzymatic ion transporters, demonstrated in electrokinetics of proton pumping rhodopsin.

H(+)-pumping rhodopsins mediate a primordial conversion of light to metabolic energy. Bacteriorhodopsin from Halobacterium salinarium is the first identified and (biochemically) best-studied H(+)-pumping rhodopsin. The electrical properties of H(+)-pumping rhodopsins, however, are known in more detail for the homolog Acetabularia rhodopsin, isolated from the eukaryotic green alga Acetabularia acetabulum. Based on data from Acetabularia rhodopsin we present a general reaction kinetic model of H(+)-pumping rhodopsins with only seven independent parameters, which fits the kinetic properties of photocurrents as functions of light, transmembrane voltage, internal and external pH, and time. The model describes fast photoisomerization of retinal with simultaneous H(+) transfer to an H(+) acceptor, reprotonation of retinal from the intracellular face via an H(+) donor, and proton release to the extracellular space via an H(+) release complex. The voltage sensitivities of the individual reaction steps and their temporal changes are treated here by a novel approach, whereby--as in an Ohmic voltage divider--the effective portions of the total transmembrane voltage decrease with the relative velocities of the individual reaction steps. This analysis quantitatively infers dynamic changes of the voltage profile and of the pK values of the H(+)-binding sites involved.

Transcriptional feedback oscillators: maybe, maybe not...

The molecular mechanism of circadian rhythmicity is usually modeled by a transcription/translation feedback oscillator in which clock proteins negatively feed back on their own transcription to produce rhythmic levels of clock protein mRNAs, which in turn cause the production of rhythmic levels of clock proteins. This mechanism has been applied to all model organisms for which molecular data are available. This review summarizes the increasing number of anomalous observations that do not fit the standard molecular mechanism for the model organisms Acetabularia, Synechococcus, Drosophila, Neurospora, and mouse. The anomalies fall into 2 classes: observations of rhythmicity in the organism when transcription of clock genes is held constant, and rhythmicity in the organism when clock gene function is missing in knockout mutants. It is concluded that the weight of anomalies is now so large that the standard transcription/translation mechanism is no longer an adequate model for circadian oscillators. Rhythmic transcription may have other functions in the circadian system, such as participating in input and output pathways and providing robustness to the oscillations. It may be most useful to think in terms of a circadian system that uses a noncircadian oscillator consisting of metabolic feedback loops, which acquires its circadian properties from additional regulatory molecules such as the products of canonical clock genes.

Prokaryotic and eukaryotic unicellular chronomics.

An impeccable time series, published in 1930, consisting of hourly observations on colony advance in a fluid culture of E. coli, was analyzed by a periodogram and power spectrum in 1961. While the original senior author had emphasized specifically periodicity with no estimate of period length, he welcomed further analyses. After consulting his technician, he knew of no environmental periodicity related to human schedules other than an hourly photography. A periodogram analysis in 1961 showed a 20.75-h period. It was emphasized that "... the circadian period disclosed is not of exactly 24-h length." Confirmations notwithstanding, a committee ruled out microbial circadian rhythms based on grounds that could have led to a different conclusion, namely first, the inability of some committee members to see (presumably by eyeballing) the rhythms in their own data, and second, what hardly follows, that there were "too many analyses" in the published papers. Our point in dealing with microbes and humans is that analyses are indispensable for quantification and for discovering a biologically novel spectrum of cyclicities, matching physical ones. The scope of circadian organization estimated in 1961 has become broader, including about 7-day, about half-yearly, about-yearly and ex-yearly and decadal periodisms, among others. Microbial circadians have become a field of their own with eyeballing, yet time-microscopy can quantify characteristics with their uncertainties and can assess broad chronomes (time structures) with features beyond circadians. As yet only suggestive differences between eukaryotes and prokaryotes further broaden the perspective and may lead to life's sites of origin and to new temporal aspects of life's development as a chronomic tree by eventual rhythm dating in ontogeny and phylogeny.

Poly(A)+ RNA and cytoskeleton during cyst formation in the cap ray of Acetabularia peniculus.

The configuration and distribution of polyadenylated RNA (poly(A)+ RNA) during cyst formation in the cap rays of Acetabularia peniculus were demonstrated by fluorescence in situ hybridization using oligo(dT) as a probe, and the spatial and functional relationships between poly(A)+ RNA and microtubules or actin filaments were examined by immunofluorescence microscopy and cytoskeletal inhibitor treatment. Poly(A)+ RNA striations were present in the cytoplasm of early cap rays and associated with longitudinal actin bundles. Cytochalasin D destroyed the actin filaments and caused a dispersal of the striations. Poly(A)+ RNA striations occurred in the cytoplasm of the cap rays up to the stage when secondary nuclei migrated into the cap rays, but they disappeared after the secondary nuclei were settled in their positions. At that time, a mass of poly(A)+ RNA was present around each of the secondary nuclei and accumulated rRNA. This mass colocalized with microtubules radiating from the surface of each secondary nucleus and disappeared when the microtubules were depolymerized by butamifos, which did not affect the configuration of actin filaments. These masses of poly(A)+ RNA continued to exist even after the cap ray cytoplasm divided into cyst domains. Thus two distinct forms of poly(A)+ RNA population, striations and masses, appear in turn at consecutive stages of cyst formation and are associated with distinct cytoskeletal elements, actin filaments and microtubules, respectively.

Differential messenger RNA gradients in the unicellular alga Acetabularia acetabulum. Role of the cytoskeleton.

The unicellular green alga Acetabularia acetabulum has proven itself to be a superior model for studies of morphogenesis because of its large size and distinctive polar morphology. The giant cell forms an elongated tube (a stalk of up to 60 mm in length), which at its apical pole makes whorls of hairs, followed by one whorl of gametophores in the shape of a cap. At its basal pole, the cell extends into a rhizoid wherein the single nucleus is positioned. In this study, we have determined the level of specific messenger RNAs in the apical, middle, and basal regions using reverse transcriptase-PCR methodology. Four mRNA classes were distinguished: those that were uniformly distributed (small subunit of Rubisco, actin-1, ADP-glucose, centrin, and alpha- and beta-tubulin), those that expressed apical/basal (calmodulin-4) or basal/apical gradients (calmodulin-2 and a Ran-G protein), and those with development-specific patterns of distribution (mitogen-activated protein kinase, actin-2, and UDP-glucose-epimerase). Restoration of the apical/basal calmodulin-4 mRNA gradient after amputation of the apical region of the cell requires the nucleus and was abolished by cytochalasin D. Accumulation of actin-1 mRNA in the vicinity of the wound set by the amputation needs, likewise, the presence of the nucleus and was also inhibited by cytochalasin. This suggests that actin microfilaments of the cytoskeleton are involved in directed transport and/or anchoring of these mRNAs.

Relationships between growth, morphology and wall stress in the stalk of Acetabularia acetabulum.

With carbon particles we analyzed patterns of growth in Acetabularia acetabulum (Lam.) P.C. Silva, a giant unicell famous for classic development studies. We focused on the stalk apex, which generates the stalk, whorls of hairs, and whorls of gametophores. To gain visual and physical accessibility, we amputated the youngest whorls of hair and the original apex and performed experiments on the apex that regenerated. Video analysis indicated that most growth occurred near the tip of the new apex. Less growth occured throughout the cut-interwhorl. We also analyzed cell wall thickness along stalks cleared of cytoplasm. Correlating growth data to wall morphology suggests growth near the apex may be proportional to stress on the cell wall. We propose that turgor-pressure wall stress modulates local apical cell wall growth rates. A supplementary model, relating cell wall curvature and growth rate in the cut-interwhorl, characterizes how the stalk's final dimensions and nearly cylindrical shap may arise. See http://faculty.washington.edu/mandoli/vondassow for supplementary data, analysis, and mathematical appendices. We believe this is the first quantiative description of apex morphogenesis of A. acetabulum.

S-(1,2-Dicarboxyethyl)glutathione in yeast: partial purification of its synthesizing enzyme.

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) was found in Saccharomyces cerevisiae, but not in bacterial species nor in a unicellular alga (Acetabularia acetabulum). The enzyme that catalyzes condensation of L-malate and glutathione (GSH) to form DCE-GS was partially purified from baker's yeast. It had a molecular mass of 49 kDa and was monomeric and the Km values were 2.2 and 1.4 mM for L-malate and GSH, respectively. The enzyme had a pH optimum of 7.5. DCE-GS levels in yeast cells were significantly higher in aerobic cultures than in anaerobic ones. DCE-GS was synthesized in cells cultured between 20 and 35 degrees C.

Exchangeability of the b subunit of the Cl(-)-translocating ATPase of Acetabularia acetabulum with the beta subunit of E. coli F1-ATPase: construction of the chimeric beta subunits and complementation studies.

The gene encoding the b subunit of the Cl(-)-translocating ATPase (aclB) was isolated from total RNA and poly(A)+ RNA of Acetabularia acetabulum and sequenced (total nucleotides of 3038 bp and an open reading frame with 478 amino acids). The deduced amino acid sequence showed high similarity to the beta subunit of the F type ATPases, but was different in the N-terminal 120 amino acids. The role of the N-terminal region was investigated using an F -ATPase beta-less mutant of E. coli, JP17. The JP17 strain expressing the aclB could not grow under conditions permitting oxidative phosphorylation, although ACLB was detected in the membrane fraction. The beta subunit was divided into three portions: amino acid position from 1 to 95 (portion A), 96 to 161 (portion B) and 162 to the C-terminus (portion C). The corresponding regions of ACLB were designated as portions A' (from 1 to 106), B' (from 107 to 172) and C' (from 173 to 478). Chimeric proteins with combinations of A-B'-C', A-B-C' and A'-B-C restored the function as the beta subunit in E. coli F0F1-complex, but those with combinations of A'-B'-C and A-B'-C had no function as the beta subunit. These findings suggested that portion B plays an important role in the assembly and function of the beta subunit in the F0F1-complex, while portion B' of ACLB exhibited inhibitory effects on assembly and function. In addition, portion A was also important for interaction of the beta subunit with the alpha subunit in E. coli F0F1-complex. These findings also suggested that the b subunit of the Cl(-)-translocating ATPase of A. acetabulum has a different function in the Cl(-)-translocating ATPase complex, although the primary structure resembled to the beta subunit of the F1-ATPase.

Decreasing period-length of the endogenous circadian rhythm of oxygen evolution in Acetabularia and its possible relation to aging.

Endogenous circadian rhythms observed under constant conditions normally show period length variations. However, a general trend is difficult to identify when cells or organisms are entrained with the usual 24-h-period light/dark cycles. Therefore, these variations in time have been considered as fluctuations. In order to gain more insight into this phenomenon, individual Acetabularia cells were exposed to light/dark cycles of 16 h (LD 8:8) and 33.6 h (LD 16.8:16.8), respectively, i.e., periods which lie distinctly outside the range of the normal circadian entrainment. Employing a high-resolution procedure for data analysis, decreasing period lengths could consistently be detected when cells were kept under constant conditions for several weeks. Possible causes of this decrease are discussed.

Na+ transport in Acetabularia bypasses conductance of plasmalemma.

Na(+)-selective microelectrodes with the sensor ETH 227 have been used to measure the cytoplasmic Na+ concentration, [Na+]c, in Acetabularia. In the steady-state, [Na+]c is about 60 mM (external 460 mM). Steps in external Na+ concentration, [Na+]o, cause biexponential relaxations of [Na+]c which have formally been described by a serial three-compartment model (outside<==>compartment 1<==>compartment 2). From the initial slopes (some mMsec-1) net uptake and release of about 3 mumolm-2sec-1 Na+ are determined. Surprisingly, but consistent with previous tracer flux measurements (Mummert, H., Gradmann, D. 1991. J. Membrane Biol, 124:255-263), these Na+ fluxes are not accompanied by corresponding changes of the transplasmalemma voltage. [Na+]c is neither affected by the membrane voltage, nor by electrochemical gradients of H+ or Cl- across the plasmalemma, nor by cytoplasmic ATP. The results suggest a powerful vesicular transport system for ions which bypasses the conductance of the plasmalemma. In addition, transient increases of [Na+]c have been observed to take place facultatively during action potentials. The exponential distribution of the amplitudes of these transients (many small and few large peaks) points to local events in the more ore less close vicinity of the Na+ recording electrode. These events are suggested to consist of disruption of endoplasmic vesicles due to a loss of pressure in the cytoplasm.

Characterization of ion channels from Acetabularia plasma membrane in planar lipid bilayers.

Plasma membrane from Acetabularia acetabulum was prepared by aqueous-polymer two-phase partitioning and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers by stirring in the presence of a (cis:trans) 325:100 mM KCl gradient. Under these conditions five distinct K(+)-selective channels were observed which had unitary chord-conductances (determined between 30 mV either side of the reversal potential) and frequencies of incorporation (in parentheses) of 1,600 pS (26%), 485 pS (21%), 259 pS (53%), 140 pS (37%) and 27 pS (37%). Two Cl(-)-selective channels were also observed, which had unitary chord-conductances of 8 and 48 pS and were present in 21 and 16% of bilayers, respectively. The voltage dependencies of channel open probability (Po), open-state time constant (tau o) and closed-state time constant (tau c) were determined for the 259, 140 and 27 pS K+ channels. The Po of all three channels increased with increasingly positive membrane potentials. Thus, since these channels were oriented with their extracellular face adjacent to the cis chamber, which was grounded, all would exhibit outward rectification in vivo. Changes in Po were effected by modulation of tau c in all channels, which shortened as membrane potentials became more positive, and also of tau o in the 140 and 27 pS channels, which increased as membrane potentials became more positive. Extracellular (cis) KCl concentration (and/or the KCl gradient across the bilayer) affected the Po of all three K+ channels, shifting the Po/membrane potential relationship in the direction of the change in the potassium reversal potential. In all channels this was achieved largely by changes in tau c.

Identification of two clock proteins in Acetabularia cliftonii and construction of cDNA libraries from Acetabularia cliftonii and Acetabularia mediterranea.

1. Two clock proteins were identified in A. cliftonii. The first has a molecular weight (mol. wt) of 200 kDa (P200) and its synthesis shows a 24 hr periodicity. The second has mol. wt of 130 kDa (P130) and shows a semicircadian rhythm with a periodicity of about 12 hr. 2. cDNA libraries from A. cliftonii and A. mediterranea were prepared by cloning cDNA in lambda gt10 and lambda gt11, respectively. 3. One clone each of the two libraries hybridized with the human beta-actin pseudogene. One clone of the A. mediterranea and 4 clones of the A. cliftonii libraries hybridized to Chlamydomonas heat-shock gene.