T he circadian clock is a molecular mechanism underlying endogenous, self-sustained oscillations with a period of Ϸ24 h, manifested in diverse physiological and metabolic processes (1-3). The most striking feature of circadian clock is its flexible yet robust response to various environmental conditions. For example, circadian periodicity varies with light intensity (4-6) while remaining robust over a wide range of temperatures (''temperature compensation'') (1, 3, 7-9). This flexible-yetrobust characteristic is evolutionarily conserved in organisms ranging from photosynthetic bacteria to warm-blooded mammals (3, 10-12), and has interested researchers from a broad range of disciplines. However, despite many genetic and molecular studies (13-22), the detailed biochemical mechanism underlying this characteristic remains poorly elucidated (3).The simplest explanation for this flexible-yet-robust property is that the key period-determining reactions are insensitive to temperature but responsive to other environmental conditions. Indeed, Pittendrigh proposed the existence of a temperatureinsensitive component in the clock system in 1954 (7), and in 1968, he and his colleagues demonstrated that both the wave form and the period of circadian oscillations are invariant with temperature (23). However, the idea of a temperatureinsensitive biochemical reaction is counterintuitive, as elementary chemical processes are highly temperature-sensitive. One exception is the cyanobacterial clock, in which temperatureinsensitive enzymatic reactions are observed (24,25). However, the cyanobacterial clock is quite distinct from other clock systems, and this biochemical mechanism has not been demonstrated in other clocks.Recently, a chemical-biological approach was proposed to help elucidate the basic processes underlying circadian clocks (26), and high-throughput screening of a large chemical compound library was performed (27). In this report, to analyze systematically the fundamental processes involved in determining the period length of mammalian clocks, we tested 1,260 pharmacologically active compounds for their effect on period length in mouse and human clock cell lines, and found 10 compounds that most markedly lengthened the period of both clock cell lines affected both the central and peripheral circadian clocks. Most compounds inhibited CKI or CKI␦ activity, suggesting that CKI /␦-dependent phosphorylation is an important period-determining process in the mammalian circadian clock. Surprisingly, the degradation rate of endogenous PER2, which is regulated by CKI -dependent phosphorylation (28) and probably by CKI␦-dependent phosphorylation, was temperatureinsensitive in the living clock cells, and the temperatureinsensitivity was preserved even for the in vitro CKI /␦-dependent phosphorylation of a synthetic peptide derived from PER2. These results suggest that this period-determining process is flexible in response to chemical perturbation yet robust in the face of temperature perturbations. Based on these findings, we prop...

Centromeric heterochromatin assembly in fission yeast requires the RNAi pathway. Chp1, a chromodomain (CD) protein, forms the Ago1-containing RNA-induced transcriptional silencing (RITS) complex and recruits siRNA-bound RITS to methylated histone H3 lysine 9 (H3K9me) via its CD. Here, we show that the CD of Chp1 (Chp1-CD) possesses unique nucleic acid-binding activities that are essential for heterochromatic gene silencing. Detailed electrophoretic-mobility shift analyses demonstrated that Chp1 binds to RNA via the CD in addition to its central RNA-recognition motif. Interestingly, robust RNA- and DNA-binding activity of Chp1-CD was strongly enhanced when it was bound to H3K9me, which was revealed to involve a positively charged domain within the Chp1-CD by structural analyses. These results demonstrate a role for the CD that provides a link between RNA, DNA, and methylated histone tails to ensure heterochromatic gene silencing.

As the SH-reactive fluorescein derivative eosin-5-maleimide (EMA) specifically labels Cys159 in the second loop facing the matrix space (loop M2) of the ADP/ATP carrier in bovine heart submitochondrial particles [Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187], we studied the interaction of non-SH-reactive eosin Y, an analog of EMA, with the carrier under various conditions to characterize its binding. Eosin Y was found to inhibit ADP transport by binding to loop M2 in submitochondrial particles, but not in mitochondria. Its Ki for transport (0.33 microM) was found to be very similar to its Kd (0.53 microM) for specific binding to the carrier. Bound eosin Y was displaced by the transport substrates ADP and ATP, but not by untransportable GTP, suggesting that eosin Y bound to the specific binding site of ADP and ATP. The three-dimensional structure and electrostatic features of eosin Y were very similar to those of ADP, and the hydrophobic property and divalent charge of eosin Y were very important for its binding to the carrier. Based on these results, the features of the binding site of the transport substrates are considered.

In fission yeast, the RNAi pathway is required for centromeric heterochromatin assembly. siRNAs derived from centromeric transcripts are incorporated into the RNA-induced transcriptional silencing (RITS) complex and direct it to nascent homologous transcripts. The RNA-induced transcriptional silencing-bound nascent transcripts further recruit the RNA-directed RNA polymerase complex (RDRC) to promote dsRNA synthesis and siRNA production. Heterochromatin coated with Swi6/Heterochromain Protein 1 is then formed following recruitment of chromatin modification machinery. Swi6 is also required for the upstream production of siRNA, although the mechanism for this has remained obscure. Here, we demonstrate that Swi6 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. An ers1 + mutant allele (ers1-C62) was identified in a genetic screen for mutants that alleviate centromeric silencing, and this phenotype was suppressed by overexpression of either the Hrr1 RDRC subunit or Clr4 histone H3-K9 methyltransferase. Ers1 physically interacts with Hrr1, and loss of Ers1 impairs RDRC centromeric localization. Although Ers1 failed to bind Clr4, a direct interaction with Swi6 was detected, and centromeric localization of Swi6 was enhanced by Clr4 overexpression in ers1-C62 cells. Consistent with this, deletion of swi6 + reduced centromeric localization of Ers1 and RDRC. Moreover, tethering of Ers1 or Hrr1 to centromeric heterochromatin partially bypassed Swi6 function. These findings demonstrate an alternative mechanism for RDRC recruitment and explain the essential role of Swi6/Heterochromain Protein 1 in RNAi-directed heterochromatin assembly.RNA interference | histone H3-lysine 9 methylation I n a eukaryotic cell, the formation of higher order chromatin structure, heterochromatin, is critical for genomic stability, chromosome segregation, and epigenetic gene silencing. Heterochromatic structure is also essential for functional organization of chromosomal domains, such as the centromeres and telomeres, and is defined by specific posttranslational modifications of nucleosome histone tails. The methylation of lysine 9 of histone H3 (H3K9me) is a key marker of heterochromatin and is provided by the methyltransferase Clr4/Suv3-9 (1-3). This H3K9me marker serves as a binding site for Heterochromatin Protein 1 (HP1) family proteins, which provide a platform for recruitment of transacting factors to maintain repressive chromatin structure (4).In the fission yeast Schizosaccharomyces pombe, the assembly of heterochromatin at centromeres also depends on transcription of the centromeric dg and dh repeats by RNA polymerase II during S phase (5-7) and the subsequent recruitment of RNAi machineries (8-10). In this RNAi system, siRNAs derived from the centromeric repeats and the RNA-induced transcriptional silencing (RITS) complex play a central role to establish H3K9me at centromeric regions (11). RITS complex, containing Argonaute (Ago1), the GW-repeat Tas3 protein, and the chromodomain (CD) protein Chp1, targets nasce...

No abstract

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys 185 in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0°C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424 -432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys 185 . In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.There are various solute carriers in the mitochondrial inner membrane to support ATP synthesis by oxidative phosphorylation. The 30-kDa solute carriers, consisting of a three-repeat structure containing a certain consensus sequence, are members of the mitochondrial solute carrier family (1). Of these, the ADP/ATP carrier mediating transport of ADP and ATP, the phosphate carrier mediating the symport of orthophosphate (P i ) and H ϩ , and the type 1 uncoupling protein forming the short circuit of the proton current (2-4) have received considerable attention. These carriers take similar topologies of six transmembrane helices with three large hydrophilic loops facing the matrix, and their homodimers are thought to be their functional units (2,3,5,6). However, their precise structural characteristics are not fully understood in relation to their transport functions.Because fluorescein derivatives have been thought to have similar structural features to those of adenine nucleotides (7, 8), they have been used as fluorescent probes in studies on the kinetics and conformational changes caused by their interactions with the adenine nucleotide binding sites of proteins such as ATPases (9 -11), NAD(P) ϩ -dependent dehydrogenases (12, 13), and kinases (7,8). In fact, we recently reported that the geometric and electronic structures of fluorescein analogs are very similar to those of ADP/ATP (14). In addition, we found that various fluorescein derivatives have high affinities to the ADP/ATP carrier in bovine heart mitochondria, and the binding leads to inhibition of the transport activity (4,14,15). Of the fluorescein analogs, the SH reagent eosin 5-maleimide (EMA) 1 most significantly interacts with the ADP/ATP carrier; it quickly and specifically labels Cys 159 in the second loop facing the matrix of the bovine ...