We describe a novel requirement for the condensin complex in sister chromatid cohesion in Saccharomyces cerevisiae. Strikingly, condensin-dependent cohesion can be distinguished from cohesin-based pairing by a number of criteria. First, condensin is required to maintain cohesion at several chromosomal arm sites but, in contrast to cohesin, is not required at either centromere or telomere-proximal loci. Second, condensin-dependent interlinks are established during mitosis independently of DNA replication and are reversible within a single cell cycle. Third, the loss of condensin-dependent linkages occurs without affecting cohesin levels at the separated URA3 locus. We propose that, during mitosis, robust sister chromatid cohesion along chromosome arms requires both condensin-and cohesin-dependent mechanisms, which function independently of each other. We discuss the implications of our results for current models of sister chromatid cohesion.

SignificanceThe ubiquitin ligase CRL4COP1/DET1 modifies specific transcription factor substrates with polyubiquitin so that they are degraded. However, the Ras–MEK–ERK signaling pathway can inactivate CRL4COP1/DET1 and thereby promote the rapid accumulation of these transcription factors. Here we show that constitutive photomorphogenesis 1 (COP1) has a critical role in mouse brain development because its deletion from neural stem cells stabilizes the transcription factors c-JUN, ETV1, ETV4, and ETV5, leading to perturbation of normal gene expression patterns; anatomic anomalies in cerebral cortex, hippocampus, and cerebellum; and perinatal lethality.

Lipid kinases have emerged as potentially important therapeutic targets in oncology and inflammation. Ceramide kinase (CERK) is a lipid kinase that catalyzes the formation of ceramide-1-phosphate from ceramide, a sphingolipid that is a key mediator of cellular apoptosis. Ceramide-1-phosphate has been shown to enhance the production of pro-inflammatory eicosonoids, to promote cell proliferation, and potentially to reduce intracellular ceramide levels by inhibition of acidic sphingomyelinases. Here we describe a homogeneous chemiluminescence assay that directly measures the ceramide-dependent ATP depletion by recombinant full-length human CERK. As compared to reported CERK assays that have limitations on compound throughput, the chemiluminescence assay has been miniaturized to a 1,536-well microtiter plate format and utilized to screen an ultra-large compound library (>4 million compounds). Multiple chemical scaffolds have been identified as CERK kinase inhibitors and characterized mechanistically, which to our knowledge represent the first known small molecule CERK inhibitors with nanomolar activities. These compounds can serve as tools to further elucidate the CERK pathway and its role in ceramide metabolism and human diseases.

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Functional flagella formation is a widespread virulence factor that plays a critical role in survival and host colonization. Flagellar synthesis is a complex and highly coordinated process. The assembly of the axial structure beyond the cell membrane is mediated by export chaperone proteins that transport their cognate substrates to the export gate complex. The export chaperone FliS interacts with flagellin, the basic component used to construct the filament. Unlike enterobacteria, the gastric pathogen Helicobacter pylori produces two different flagellins, FlaA and FlaB, which exhibit distinct spatial localization patterns in the filament. Previously, we demonstrated a molecular interaction between FliS and an uncharacterized protein, HP1076, in H. pylori. Here, we present the crystal structure of FliS in complex with both the C-terminal D0 domain of FlaB and HP1076. Although this ternary complex reveals that FliS interacts with flagellin using a conserved binding mode demonstrated previously in Aquifex aeolicus, Bacillus subtilis, and Salmonella enterica serovar Typhimurium, the helical conformation of FlaB in this complex was different. Moreover, HP1076 and the D1 domain of flagellin share structural similarity and interact with the same binding interface on FliS. This observation was further validated through competitive pull-down assays and kinetic binding analyses. Interestingly, we did not observe any detrimental flagellation or motility phenotypes in an hp1076-null strain. Our localization studies suggest that HP1076 is a membraneassociated protein with a cellular localization independent of FliS. As HP1076 is uniquely expressed in H. pylori and related species, we propose that this protein may contribute to the divergence of the flagellar system, although its relationship with FliS remains incompletely elucidated.