PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein-Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.

Cancer cells are critically dependent on ER–mitochondria Ca2+ flux that regulates their bioenergetics. Here, Raturi et al. identify the ER oxidoreductase TMX1 as a thiol-dependent regulator of this intracellular signaling mechanism within cancer cells.

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA-and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNAactivated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs. INTRODUCTIONThe Argonaute superfamily comprises a group of RNAbinding proteins that form the cores of ribonucleoprotein complexes (RNPs) that mediate RNA interference (RNAi) and related gene-silencing pathways (reviewed in Hannon, 2002;Peters and Meister, 2007;Hutvagner and Simard, 2008). All metazoans encode multiple Argonaute genes, the products of which have unique as well as overlapping functions. The basic functions of Argonautes in the canonical RNAi pathway are reasonably well understood. Small guide RNAs direct Argonaute-containing RNPs to homologous mRNAs, thereby providing specificity in this pathway (reviewed in Hannon, 2002;Jaronczyk et al., 2005). The Argonaute superfamily has been divided into two subgroups, Argonaute and Piwi (Bohmert et al., 1998;Cox et al., 1998;Carmell et al., 2002). Expression of the Piwi subfamily members is restricted to germline tissues and undifferentiated cells, whereas the Argonaute group members are ubiquitous. The two main types of small RNAs that associate with Argonaute subfamily members differ in origin and function. Short interfering RNAs (siRNAs) and microRNAs (miRNAs) are derived from long double-stranded (ds)RNA and hairpin RNA precursors, respectively. Successive processing of miRNA precursors in the nucleus and cytoplasm, respectively, by the RNases Drosha (Lee et al., 2003) and Dicer (Bernstein et al., 2001) produces mature miRNAs that are then transferred to an Argonaute-containing RNP complex termed RNA induced silencing complex (RISC) (Hammond et al., 2000). The biogenesis of siRNAs only requires Dicer activity. A third species of small noncoding RNAs (Piwiinteracting RNAs) that associate with Piwi subfamily members was recently discovered (Aravin et al., 2006;Girard et al., 2006). However, their expression is limited to germline tissues in which they are thought to function in gametogenesis and retrotransposon silen...

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998;Cox et al., 1998;Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes. INTRODUCTIONThe cytoplasm of eukaryotic cells is partitioned into more than a dozen membrane-bound organelles. Compartmentalization serves to increase the efficiencies of cellular processes by controlling the spatial and temporal interactions of proteins, nucleic acids and lipids. The endoplasmic reticulum (ER) and Golgi complex play central roles in the biogenesis and operational fidelity of eukaryotic cells by orchestrating the synthesis and movement of proteins and lipids (Hurtley and Helenius, 1989;Narula et al., 1992;Sitia and Meldolesi, 1992;Bergeron et al., 1994;Hammond and Helenius, 1995;Farquhar and Hauri, 1997;Hauri and Schweizer, 1997;Farquhar and Palade, 1998). It is now clear that these two organelles are directly involved in processes that affect cellular differentiation because mistargeting and/or altered expression of resident proteins of the ER and Golgi complex can have profound effects on cell growth, morphology, and tumorigenicity. Moreover, cellular defects at the ER/Golgi level underlie the pathophysiology of many human diseases such as familial hypercholesterolemia, polycystic kidney disease, Tangier disease, cystic fibrosis, mucopolysaccharidosis types I, VI, and VII, progeroid syndrome, and many others (for review, see Brooks, 1997;Gonatas, 1997).We are interested in identifying novel ER-and Golgiassociated proteins that are involved in the function of these organelles. The approach we adopted was to generate a panel of mAbs against ER/Golgi membrane fractions so that they may be used to identify and then immunoaffinity purify the...

GERp95 (Golgi-endoplasmic reticulum protein 95 kDa) is part of a large family of highly conserved proteins found in all metazoans and the fission yeast Schizosaccharomyces pombe. Genetic studies suggest that homologs of GERp95 are components of signaling pathways that regulate cellular differentiation, development, and RNA interference. However, the precise molecular functions of these proteins remain unknown. Genetic analysis of GERp95 homologs has been complicated by the presence of multiple genes with overlapping functions in most organisms. Binding partners for members of this protein family have not been identified. The purpose of this study was to identify proteins that associate with GERp95. Glutathione S-transferaseGERp95 fusions were expressed in transfected cells, and proteins that bound to GERp95 were co-purified using glutathione-agarose beads. The amino-terminal region of GERp95 was found to interact with the specialized chaperone Hsp90 and a number of its cognate binding proteins. Inhibition of Hsp90 activity with geldanamycin or radicicol resulted in rapid degradation of newly synthesized GERp95. The membrane-associated pool of GERp95 was not bound to Hsp90, although activity of this chaperone was required for stable association of GERp95 with the Golgi in normal rat kidney cells. These results indicate that GERp95 engages an Hsp90 chaperone complex prior to association with intracellular membranes.