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following correction should be noted. Due to an editorial change at PNAS, the meaning of the last sentence on page 14046 was altered. The sentence originally read as follows: On the other hand, this structure does not reproduce the pharmacological properties of either P or Q channel exactly, as the ID 50 to sFTX and -Aga IVA for P-type channels is lower than for the ␣1A, ␣2␦, ␤Ib channels in HEK cells.Neurobiology. In the article "The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide" Kuo Wu, Chiye Aoki, Alice Elste, Adrienne A. Rogalski-Wilk, and Philip Siekevitz, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(13273)(13274)(13275)(13276)(13277)(13278), the quality of the reproduction of Fig. 2A was poor. The figure and its legend are shown below:Biochemistry. In the article "KSR stimulates Raf-1 activity in a kinase-independent manner" by Neil R. Michaud, Marc Therrien, Angela Cacace, Lisa C. Edsall, Sarah Spiegel, Gerald M. Rubin, and Deborah K. Morrison, which appeared in number 24, November 25, 1997, of Proc. Natl. Acad. Sci. USA (94,(12792)(12793)(12794)(12795)(12796), the following correction should be noted.Due to a printer's error, background was incorrectly added to (50 g) and 100 g each of the other fractions, in 100 l final volume, including whole homogenate (H), synaptosomes (Syn), synaptic plasma membranes (SPM), and crude synaptic vesicles (CSV), were incubated at 37°C for 15 min. NAD incorporation was performed in the absence (Ϫ) or presence (ϩ) of SNP as exogenous source of NO. The mixtures were subjected to SDS͞PAGE and then autoradiography. (B) Western blot analysis of the G3PD in the subcellular fractions. To confirm that the radioactive protein in the subcellular fractions was indeed G3PD, Western blot analysis was performed by using specific anti-G3PD antibodies as described.

The plant photoreceptor phytochrome plays an important role in the nucleus as a regulator of transcription. Numerous studies imply, however, that phytochromes in both higher and lower plants mediate physiological reactions within the cytoplasm. In particular, the tip cells of moss protonemal filaments use phytochrome to sense light direction, requiring a signaling system that transmits the directional information directly to the microfilaments that direct tip growth. In this work we describe four canonical phytochrome genes in the model moss species Physcomitrella patens, each of which was successfully targeted via homologous recombination and the distinct physiological functions of each gene product thereby identified. One homolog in particular mediates positive phototropism, polarotropism, and chloroplast movement in polarized light. This photoreceptor thus interacts with a cytoplasmic signal͞response system. This is our first step in elucidating the cytoplasmic signaling function of phytochrome at the molecular level.

The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host.In the present study, His 6 -tagged Cph1 was isolated from Synechocystis cells. The cph1±his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn 21 -induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.Keywords: phytochrome; cyanobacteria; Synechocystis; chromophore; phycocyanobilin.Light plays a crucial role in the life of cyanobacteria. It provides the energy source for photosynthesis, triggers short and long-term adaptive responses of the photosyntheic apparatus [1], synchronizes circadian rhythmicity [2], acts as an attractant in phototaxis [3,4], and regulates differentiation into hormogonia and heterocysts [5,6]. However, until recently, the existence of receptors for light have remained obscure in cyanobacteria. By the analysis of sequence data available now from the unicellular cyanobacterium Synechocystis sp. PCC 6803 [7], from the filamentous cyanobacterium Anabeana sp. PCC 7120 (http:// www.kazusa.or.jp/cyano/anabaena/) and from other cyanobacterial strains [8,9] it has become obvious that cyanobacterial DNAs encode putative photoreceptors of the phytochrome-type.In plants, the phytochromes comprise a small family of homologous photoactive proteins. Triggered by red or farred light, they mediate a great number of photoresponses ranging from chloroplast rotation in green algae to seed germination, de-etiolation and control of flowering in seed plants. Plant phytochromes acquire their photoactivity upon attachment to the linear tetrapyrrole chromophores phytochromobilin [10±12] or phycocyanobilin [13]. The holoproteins are interconvertible between two thermostable, spectrally distinct forms, the red-light-absorbing form Pr, and the far-red-light-absorbing form Pfr [14]. Red light (650±660 nm) establishes a high Pfr level, whereas under far-red light (700±730 nm) Pr is formed.A similar photoactivity has recently been observed with the product of the phytochrome gene cph1 from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (henceforth referred to as Synechocystis 6803). Upon expression in Escherichia coli or yeast, Cph1 attaches linear tetrapyrrole chromophores autocatalytically, and is photointerconvertible between a Pr and a Pfr form [15±18].The deduced Cph1 protein possesse...

Specific 3'-sulfogalactolipid [SGL-sulfogalactosyl ceramide (SGCer) and sulfogalactosylglycerolipid (SGG)] binding is compared for hsp70s cloned from Helicobacter pylori, Haemophilus influenzae, Chlamydia trachomatis serovar E, Escherichia coli, murine male germ cells, and the hsp70-like extracellular domain within the sperm receptor from Strongylocentrotus purpuratus. This lectin activity, conserved among the different hsp70 family members, is modulated by the SGL aglycone. This is shown by differential binding to both SGC fatty acid homologues and 3'-sulfogalactolipid neoglycoproteins generated by coupling bovine serum albumin (BSA) and glycosyl ceramide acids synthesized by oxidation of the double bond of sphingosine. Eukaryotic hsp70s preferentially bound the SGCer fatty acid homologues SG(24)Cer, SG(18)Cer, and SG(20:OH)Cer, while prokaryotic hsp70s bound SG(18:1)Cer and SG(20:OH)Cer. Eukaryotic hsp70s bound SGCer-BSA and SG(24)Cer-BSA conjugates where the latter is the main constituent in SGCer-BSA, while prokaryotic hsp70s bound SG(20:OH)Cer-BSA. None of the hsp70s bound sulfogalactosyl sphingosine (SGSph) or SGSph-BSA, further demonstrating the important role of the aglycone. Although the primary SGL recognition domain of all hsp70s is conserved, we propose that aglycone organization differentially influences the interaction with the sub-site. Heterogeneous SGCer aglycone isoforms in cells and the differential in vitro binding of eukaryotic and prokaryotic hsp70s may relate to their different adhesin roles in vivo as mediators of germ cell and bacterial/host interactions, respectively.

Protonemata of the moss Ceratodon purpureus cultured in white light were transferred to darkness for 3 days and then used for phototropic experiments. Irradiation of the apical region of vertically position protonemata with small beams (0.2 mm) of red light induced a growth response towards the irradiated side (positive phototropism). The phototropic response showed irradiance dependence. The effect of red light was completely reversed by far-red light following red light irradiations, demonstrating that phytochrome was the photoreceptor pigment. Far-red light or UV-blue light had no influence on either bulging or phototropism. Experiments with linearly polarized red or far-red light showed a different dichroic distribution of phytochrome in its different forms, the red-absorbing form, P, and the far-red-absorbing form, Pfr. Red light with a vibration plane parallel to the long axis of the filaments was most effective. The effectiveness of far-red light was expressed best when its vibration plane was 90" to the electrical vector of the inductive red light.

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