The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.

The endoplasmic reticulum (ER) plays an essential role in the production of lipids and secretory proteins. Because the ER cannot be generated de novo, it must be faithfully transmitted or divided at each cell division. Little is known of how cells monitor the functionality of the ER during the cell cycle or how this regulates inheritance. We report here that ER stress in S. cerevisiae activates the MAP kinase Slt2 in a new ER Stress Surveillance (ERSU) pathway, independent of the Unfolded Protein Response. Upon ER stress, ERSU alters the septin complex to delay ER inheritance and cytokinesis. In the absence of Slt2 kinase, the stressed ER is transmitted to the daughter cell, causing the death of both mother and daughter cells. Furthermore, Slt2 is activated via the cell surface receptor Wsc1 by a previously undescribed mechanism. We conclude that the novel ERSU pathway ensures inheritance of a functional ER.

Although introns in 5 0 -and 3 0 -untranslated regions (UTRs) are found in many protein coding genes, rarely are they considered distinctive entities with specific functions. Indeed, mammalian transcripts with 3 0 -UTR introns are often assumed nonfunctional because they are subject to elimination by nonsense-mediated decay (NMD). Nonetheless, recent findings indicate that 5 0 -and 3 0 -UTR intron status is of significant functional consequence for the regulation of mammalian genes. Therefore these features should be ignored no longer.

When unfolded proteins accumulate in the endoplasmic reticulum (ER) causing ER stress, the unfolded protein response (UPR) responds rapidly to induce a transcriptional program that functions to alleviate the stress. However, under extreme conditions, when UPR activation is not sufficient to alleviate ER stress, the stress may persist long term. Very little is known about how the cell responds to persistent ER stress that is not resolved by the immediate activation of the UPR. We show that Hog1 MAP kinase becomes phosphorylated during the late stage of ER stress and helps the ER regain homeostasis. Although Hog1 is well known to function in osmotic stress and cell wall integrity pathways, we show that the activation mechanism for Hog1 during ER stress is distinct from both of these pathways. During late stage ER stress, upon phosphorylation, Hog1 translocates into the nucleus and regulates gene expression. Subsequently, Hog1 returns to the cytoplasm, where its phosphorylation levels remain high. From its cytoplasmic location, Hog1 contributes to the activation of autophagy by enhancing the stability of Atg8, a critical autophagy protein. Thus, Hog1 coordinates a multifaceted response to persistent ER stress.

The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0 -12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx⅐DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx⅐DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNAbinding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either singlestranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated.

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of Tand B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4 ؉ CD8 ؉ double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4 ؉ T-cell population but was active in the CD8؉ cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.Pluripotent stem cells are constantly faced with critical decisions between self-renewal and starting to differentiate into various cell types (1, 2). Commitment of differentiating stem cells toward the various lineages is influenced by many factors, including microenvironment and external cues that are integrated into signaling pathways regulating transcriptional programs and protein production. Hematopoietic stem cells (HSC) 3 that differentiate into the erythroid, myeloid, or lymphoid lineages, primarily in the bone marrow of vertebrate adults, undergo dramatic changes in cellular architecture during differentiation to functionally specialized cells. For cells that are specialized for protein secretion, such changes include the creation of extraordinary protein processing and secretory apparatus. The unfolded protein response (UPR) is a conserved signal transduction pathway that in response to endoplasmic reticulum (ER) stress enables cells to increase the protein folding capacity of the ER, the major cellular compartment for folding and maturation of secreted and membrane proteins, by increasing the transcription of genes involved in protein folding. In addition, UPR activation also increases expression of genes involved in ER membrane biosynthesis, presumably resulting in ER expansion. Thus, UPR signaling may play a role in the differentiation of HSC. In fact, an importance for the UPR signaling components IRE1 and XBP1 has been demonstrated during the terminal differentiation of activated B lymphocytes (B cells) to immunoglobulin-secreting plasma cells (3-6).In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, serve to monitor ER protein folding needs and initiate UPR activation (7-10). In additio...

The unfolded protein response (UPR) pathway helps cells cope with endoplasmic reticulum (ER) stress by activating genes that increase the ER's functional capabilities. We have identified a novel role for the UPR pathway in facilitating budding yeast cytokinesis. Although other cell cycle events are unaffected by conditions that disrupt ER function, cytokinesis is sensitive to these conditions. Moreover, efficient cytokinesis requires the UPR pathway even during unstressed growth conditions. UPR-deficient cells are defective in cytokinesis, and cytokinesis mutants activate the UPR. The UPR likely achieves its role in cytokinesis by sensing small changes in ER load and making according changes in ER capacity. We propose that cytokinesis is one of many cellular events that require a subtle increase in ER function and that the UPR pathway has a previously uncharacterized housekeeping role in maintaining ER plasticity during normal cell growth.

Messenger RNA (mRNA) translation and mRNA degradation are important determinants of protein output, and they are interconnected. Previously, it was thought that translation of an mRNA, as a rule, prevents its degradation. mRNA surveillance mechanisms, which degrade mRNAs as a consequence of their translation, were considered to be exceptions to this rule. Recently, however, it has become clear that many mRNAs are degraded co-translationally, and it has emerged that codon choice, by influencing the rate of ribosome elongation, affects the rate of mRNA decay. In this review, we discuss the links between translation and mRNA stability, with an emphasis on emerging data suggesting that codon optimality may regulate mRNA degradation.