In Drosophila, the MSL complex is required for the dosage compensation of X-linked genes in males and contains a histone acetyltransferase, MOF. A point mutation in the MOF acetyl-CoA-binding site results in male-specific lethality. Yeast Esa1p, a MOF homolog, is essential for cell cycle progression and is the catalytic subunit of the NuA4 acetyltransferase complex. Here we report that NuA4 purified from yeast with a point mutation in the acetyl-CoA-binding domain of Esa1p exhibits a strong decrease in histone acetyltransferase activity, yet has no effect on growth. We demonstrate that Eaf3p (Esa1p-associated factor-3 protein), a yeast protein homologous to the Drosophila dosage compensation protein MSL3, is also a stable component of the NuA4 complex. Unlike other subunits of the complex, it is not essential, and the deletion mutant has no growth phenotype. NuA4 purified from the mutant strain has a decreased apparent molecular mass, but retains wildtype levels of histone H4 acetyltransferase activity. The EAF3 deletion and the ESA1 mutation lead to a decrease in PHO5 gene expression; the EAF3 deletion also significantly reduces HIS4 and TRP4 expressions. These results, together with those previously obtained with both the MSL and NuA4 complexes, underscore the importance of targeted histone H4 acetylation for the genespecific activation of transcription.Transcription activation has long been known to involve gene-specific factors that mediate the association of the RNA polymerase preinitiation complex with promoter regions and that induce the initiation of transcript synthesis. In recent years, numerous studies have demonstrated the occurrence of another layer of eukaryotic transcription activation mediated by protein complexes that alter chromatin conformation. Components of these complexes are conserved, underscoring the importance of their regulatory role (reviewed in Ref. 1). Chromatin-remodeling complexes can be grouped into two broad categories: those that use the energy of ATP hydrolysis to alter nucleosomal conformation (e.g. SWI/SNF in yeast, Drosophila, and mammals; RSC, ISW1, and ISW2 in yeast; NURF, CH-RAC, and ACF in Drosophila; and RSF and WCRF in mammals) and those that are recruited to alter chromatin conformation via the acetylation of histones (e.g. NuA4, 1 NuA3, ADA, Elongator, and SAGA in yeast and TAFII250, human GCN5, p/CAF, SRC-1/ACTR, and CBP/p300 complexes in mammals) or their deacetylation (reviewed in Refs. 2 and 3; see Refs. 4 and 5). The mechanisms whereby the enzymatic functions identified with these complexes are translated into the modulation of transcription still remain largely unresolved. Significant insights into the functional aspects of chromatin remodeling can be garnered by comparing complexes that share homologous components in different model systems where specific biochemical and genetic tools are available. We have begun such an analysis by studying two complexes with homologous protein subunits, the dosage compensation or MSL complex of Drosophila melanogaster and t...

The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.

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Site-directed mutagenesis was performed with the chromophore-bearing N-terminal domain of oat phytochrome A apoprotein (amino acid residues 1±595). Except for Trp366, which was replaced by Phe (W366F), all the residues exchanged are in close proximity to the chromophore-binding Cys321 (i.e. P318A, P318K, H319L, S320K, H322L and the double mutant L323R/Q324D). The mutants were characterized by their absorption maxima, and the kinetics of chromophore-binding and the P r 3P fr conversion. The strongest effect of mutation on the chromoprotein assembly, leading to an almost complete loss of the chromophore binding capability, was found for the exchanges of His322 by Leu (H322L) and Pro318 by Lys (P318K), whereas a corresponding alanine mutant (P318A) showed wild-type behavior. The second histidine (H319) is also involved in chromophore fixation, as indicated by a slower assembly rate upon mutation (H319L). For the other mutants, an assembly process very similar to that of the wild-type protein was found. The light-induced P r 3P fr conversion kinetics is altered in the mutations H319L and S320K and in the double mutant L323R/Q324D, all of which exhibited a significantly faster I 700 decay and accelerated P fr formation. P318 is also involved in the P r 3P fr conversion, the millisecond steps (formation of P fr ) being significantly slower for P318A. Lacking sufficient amounts of W366F, assembly kinetics could not be determined in this case, while the fully assembled mutant underwent the P r 3P fr conversion with kinetics similar to wild-type protein.Keywords: assembly; chromophore±protein interaction; heterologous expression; mutant; recombinant phytochrome.The phytochromes comprise a small protein family of ubiquitous photoreceptors in plants. They are activated upon light absorption by a covalently bound chromophore, the linear tetrapyrrole phytochromobilin (PFB) [1,2]. After the primary Z3E photoisomerization of the 15,16 double bond, a series of conformational changes of chromophore and protein convert the parent, physiologically inactive state (red-absorbing phytochrome, P r , l max = 667 nm) within milliseconds into the active far red-absorbing phytochrome (P fr ; l max = 730 nm) [3]. Both the photochemical reaction and the subsequent thermal processes of the chromophore are tuned by the protein environment. However, no three-dimensional structure of the protein is available which would allow exact identification of these chromophore±protein interactions. As alternative methods, variations in the chromophore structure [4±6] and site-directed mutagenesis (SDM) of the protein moiety have been employed. In view of the lack of information abouth the protein structure, SDM should preferably be confined to positions close to the chromophore-binding cysteine residue.[The position of the chromophore attachment in the presently best characterized phytochrome, phyA from oat (which is also dealt with here) is Cys321.] Mutagenesis has already revealed a strong influence of certain amino acid residues on the chromoprotein a...