Thus, the and cells formed much brighter colonies and grew healthier than or cells on the adenine-limiting (Fig.?1a) and adenine-lacking (Additional file 1: Fig. domains are drawn as boxes according to the CATH-Gene3D, Pfam, and Prosite classifications. Predicted localization signals are shown as in Additional file 1: Figure S1. Predicted trans-membrane helix regions (TMhelix) are indicated as gray boxes. The DnaJ domains belong to the CATH superfamily 1.10.287.110. Type-I Hsp40 proteins are characterized by C-terminal (purple) and central (pink) domains that belong to the CATH superfamilies 2.60.260.20 Mouse monoclonal antibody to ATIC. This gene encodes a bifunctional protein that catalyzes the last two steps of the de novo purinebiosynthetic pathway. The N-terminal domain has phosphoribosylaminoimidazolecarboxamideformyltransferase activity, and the C-terminal domain has IMP cyclohydrolase activity. Amutation in this gene results in AICA-ribosiduria and 220.127.116.11, respectively. The type-II Hsp40 protein Psi1 also contains the C-terminal domain but lacks the central domain. The other proteins are classified as the type-III Hsp40 proteins. Figure S3. Fission yeast type-I and -II Hsp40 proteins and their homologs. (A) Phylogenic tree of Hsp40 proteins. Scale-bar unit indicates the number of amino acid substitutions per site. Names of proteins are depicted as in Additional file 1: Figure S1. (B) Domain structure of Hsp40 proteins. Protein names are depicted as in (A) and amino acid lengths are shown. Protein domains and predicted localization signals are drawn as in Additional file 1: Figure S2. Locations of proteins in the cell are indicated. For clarity, Hsp40 proteins other than Droj2 are omitted. Figure S4. Schematic representation of marker integration sites. The ((marker gene. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (= 3). (C) Strand-specific RT-qPCR for the and genes located in the endogenous loci (= 3). **transcript from the mating-type locus. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (= 6). **and cells. Two-fold serially diluted whole-cell extracts were separated by SDS-PAGE followed by western blotting and Coomassie brilliant blue staining. The membrane for detecting FLAG-Ago1 with an antibody against FLAG epitope was reprobed with an antibody against -tubulin. Figure S9. mRNA expression levels of and genes. Strand-specific RT-qPCR for the and genes. Values are normalized to that of the sense strand of ribosomal 28S RNA, and are presented as means + SD (= 6). *deletion, four independently constructed clones were tested. Figure S11. Colony colors of alleles in the endogenous locus. Cells were streaked on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30C for four days. Strains with genetic background formed darker colonies. Note that the colony colors of canonical heterochromatin mutants (i.e. background were faint pink, and are not as white as those of strains with the native locus. Figure S12. Reintroduction of the R33C mutation phenocopied the original isolate. (A) Schematic diagram showing the introduction of the mutation (R33C) found in the original isolate. (B) Cells were streaked on normal YES plates (YES) and YES containing limited amount of adenine (Low adenine), and incubated at 30C for four days. In HKM-1565 and HKM-1618, the G418-resistant cassette was located upstream of the promoter. Both strains were generated by introducing as a model organism and investigated the roles of Hsp90 and Mas5 (a nucleocytoplasmic type-I Hsp40 protein) in RNAi-dependent heterochromatin assembly. Results Using a genetic screen and biochemical analyses, we identified Hsp90 and Mas5 as novel silencing factors. Mutations in the genes encoding these factors caused derepression of silencing at the pericentromere, where heterochromatin is assembled in an RNAi-dependent manner, but not at the subtelomere, where RNAi is dispensable. The mutations also caused a substantial IMD 0354 reduction in the level of dimethylation of histone H3 at Lys9 at the pericentromere, where association of the Argonaute protein Ago1 was also abrogated. Consistently, siRNA corresponding to the pericentromeric repeats was undetectable in these mutant cells. In addition, levels of Tas3, which is a protein in the RNA-induced transcriptional silencing complex along with Ago1, were reduced in the absence of Mas5. Conclusions Our results suggest that the IMD 0354 Hsps Hsp90 and Mas5 contribute to RNAi-dependent heterochromatin assembly. In particular, Mas5 appears IMD 0354 to be required to stabilize Tas3 in vivo. We infer that impairment of Hsp90 and Hsp40 also may affect the integrity of.