Intrauterine insemination or intracervical insemination with cryopreserved donor sperm in the natural cycle: a cohort study.

STUDY QUESTION: Does intrauterine insemination in the natural cycle lead to better pregnancy rates than intracervical insemination (ICI) in the natural cycle in women undergoing artificial insemination with cryopreserved donor sperm. SUMMARY ANSWER: In a large cohort of women undergoing artificial insemination with cryopreserved donor sperm, there was no substantial beneficial effect of IUI in the natural cycle over ICI in the natural cycle. WHAT IS KNOWN ALREADY: At present, there are no studies comparing IUI in the natural cycle versus ICI in the natural cycle in women undergoing artificial insemination with cryopreserved donor sperm. STUDY DESIGN, SIZE, DURATION: We performed a retrospective cohort study among all eight sperm banks in the Netherlands. We included all women who underwent artificial insemination with cryopreserved donor sperm in the natural cycle between January 2009 and December 2010. We compared time to ongoing pregnancy in the first six cycles of IUI and ICI, after which controlled ovarian stimulation was commenced. Ongoing pregnancy rates (OPRs) over time were compared using life tables. A Cox proportional hazard model was used to compare the chances of reaching an ongoing pregnancy after IUI or ICI adjusted for female age and indication. PARTICIPANTS/MATERIALS, SETTING, METHODS: We included 1843 women; 1163 women underwent 4269 cycles of IUI and 680 women underwent 2345 cycles of ICI with cryopreserved donor sperm. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were equally distributed (mean age 34.0 years for the IUI group versus 33.8 years for the ICI group), while in the IUI group, there were more lesbian women than in the ICI group (40.6% for IUI compared with 31.8% for ICI). Cumulative OPRs up to six treatment cycles were 40.5% for IUI and 37.9% for ICI. This corresponds with a hazard rate ratio of 1.02 [95% confidence interval (CI) 0.84-1.23] after controlling for female age and indication. Increasing female age was associated with a lower OPR, in both the IUI and ICI groups with a hazard ratio for ongoing pregnancy of 0.94 per year (95% CI 0.93-0.97). LIMITATIONS, REASONS FOR CAUTION: This study is prone to selection bias due to its retrospective nature. As potential confounders such as parity and duration of subfertility were not registered, the effect of these potential confounders could not be evaluated. WIDER IMPLICATIONS OF THE FINDINGS: In women inseminated with cryopreserved donor sperm in the natural cycle, we found no substantial benefit of IUI over ICI. A randomized controlled trial with economic analysis alongside, it is needed to allow a more definitive conclusion on the cost-effectiveness of insemination with cryopreserved donor sperm. STUDY FUNDING/COMPETING INTERESTS: No funding was used and no conflicts of interest are declared.

Routine Chlamydia antibody testing is of limited use in subfertile women with anovulation.

The Chlamydia antibody titre (CAT) is a test used to identify subfertile couples at increased risk for tubal pathology. The usefulness of the routine performance of CAT was evaluated in a multicentre prospective cohort study, in women without regular ovulation. Consecutive couples presenting with subfertility due to an irregular menstrual cycle or amenorrhoea were included. A total of 711 women were studied, all of whom underwent CAT. Tubal status was verified in 190 of these women. Two-sided tubal pathology was found in 5% of these women, and one-sided occlusion in 10%. Of all the women in the study group, 33 (4.6%) had an abnormal CAT, of which 21 underwent further tubal testing. Tubal pathology was found in two (10%) of these 21 patients. The sensitivity and specificity of CAT were respectively 20% and 89%. Correction for verification bias increased the specificity to 96% with a drop of the sensitivity to 9%. In subfertile couples with anovulation, the performance of CAT is not useful. It is proposed that testing for tubal disease in these women is delayed until treatment with clomiphene citrate has failed.

Degradation of conjugated linoleic acid isomers in the yeast Saccharomyces cerevisiae.

Propagation of Saccharomyces cerevisiae cells in conjugated linoleic acid (CLA) medium resulted in activation of the transcriptional machinery that responds to fatty acids. Cells utilized efficiently trans-10,cis-12 CLA, but not the corresponding cis-9,trans-11 isomer, probably due to the formation of cis-3,trans-5-dienoyl-CoA intermediates that are recalcitrant to beta-oxidation.

Saccharomyces cerevisiae Adr1p governs fatty acid beta-oxidation and peroxisome proliferation by regulating POX1 and PEX11.

Saccharomyces cerevisiae Adr1p is essential for fatty acid degradation and peroxisome proliferation. Here, the role of Adr1p was examined with respect to the transcriptional regulation of the Pip2p-Oaf1p dependent genes POX1 and PEX11. POX1 encodes the rate-limiting enzyme of peroxisomal beta-oxidation, acyl-CoA oxidase. The POX1 promoter was shown to contain a canonical Adr1p element (UAS1), within which the oleate response element (ORE) was nested. PEX11 codes for a peroxin that is critical for normal peroxisome proliferation, and its promoter was shown similarly to contain a UAS1-like element overlapping the ORE. Northern analysis demonstrated that transcriptional up-regulation of both POX1 and PEX11 was abolished in adr1 Delta mutant cells, and immunoblotting confirmed that the abundance of their gene products was dramatically reduced. Studies of an overlapping ORE/UAS1 arrangement in the CTA1 promoter revealed synergy between these elements. We conclude that overlapping ORE and UAS1 elements in conjunction with their binding factors Pip2p-Oaf1p and Adr1p coordinate the carbon flux through beta-oxidation with peroxisome proliferation.

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast.

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.

Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast.

Rck2, a yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, requires phosphorylation for activation. We provide evidence that in budding yeast, this step can be executed by the osmostress-activated mitogen-activated protein kinase Hog1. Rck2 phosphorylation was transiently increased during osmostress or in mutants with a hyperactive high osmolarity glycerol (HOG) pathway. This modification depended on catalytically active Hog1 kinase and two putative mitogen-activated protein kinase phosphorylation sites in Rck2. Immunokinase assays showed that Hog1 can directly phosphorylate Rck2 to stimulate its enzymatic activity toward translation elongation factor 2. We demonstrate that Hog1 and Rck2 are necessary for attenuation of protein synthesis in response to osmotic challenge and show that modification of elongation factor 2 induced by osmostress depends on Rck2 and Hog1 in vivo. Therefore, we propose that the transient down-regulation of protein synthesis after osmotic shock is a response not to damage but to an extracellular signal mediated by Hog1 and Rck2.

Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p.

The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3). Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated. They overlapped in the region of amino acids 318-550. Subsequent deletion analysis of Sla2p showed that amino acids 318-373 were essential for the two-hybrid protein-protein interaction. The N-terminal part of Rpg1p (aa 1-615) was essential and sufficient for the Rpg1p-Sla2p interaction. A coimmunoprecipitation assay provided additional evidence for the physical interaction of Rpg1p/Tif32p with Sla2p in vivo. Using immunofluorescence microscopy, Rpg1p and Sla2p proteins were colocalized at the patch associated with the tip of emerging bud. Considering the essential role of Rpg1p as the large subunit of the eIF3 core complex and the association of Sla2p with the actin cytoskeleton, a putative role of the Rpg1p-Sla2p interaction in localized translation is discussed.

Nucleocytoplasmic distribution of budding yeast protein kinase A regulatory subunit Bcy1 requires Zds1 and is regulated by Yak1-dependent phosphorylation of its targeting domain.

In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner.

Peroxisomal degradation of trans-unsaturated fatty acids in the yeast Saccharomyces cerevisiae.

Degradation of trans-unsaturated fatty acids was studied in the yeast Saccharomyces cerevisiae. Propagation of yeast cells on trans-9 elaidic acid medium resulted in transcriptional up-regulation of the SPS19 gene, whose promoter contains an oleate response element. This up-regulation depended on the Pip2p-Oaf1p transcription factor and was accompanied by induction of import-competent peroxisomes. Utilization of trans fatty acids as a single carbon and energy source was evaluated by monitoring the formation of clear zones around cell growth on turbid media containing fatty acids dispersed with Tween 80. For metabolizing odd-numbered trans double bonds, cells required the beta-oxidation auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase Eci1p. Metabolism of the corresponding even-numbered double bonds proceeded in the absence of Sps19p (2,4-dienoyl-CoA reductase) and Dci1p (Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase). trans-2,trans-4-Dienoyl-CoAs could enter beta-oxidation directly via Fox2p (2-enoyl-CoA hydratase 2 and d-specific 3-hydroxyacyl-CoA dehydrogenase) without the involvement of Sps19p, whereas trans-2,cis-4-dienoyl-CoAs could not. This reductase-independent metabolism of trans-2,trans-4-dienoyl-CoAs resembled the situation postulated for mammalian mitochondria in which oleic acid is degraded through a di-isomerase-dependent pathway. In this hypothetical process, trans-2,trans-4-dienoyl-CoA metabolites are generated by Delta(3)-Delta(2)-enoyl-CoA isomerase and Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and are degraded by 2-enoyl-CoA hydratase 1 in the absence of 2,4-dienoyl-CoA reductase. Growth of a yeast fox2sps19Delta mutant in which Fox2p was exchanged with rat peroxisomal multifunctional enzyme type 1 on trans-9,trans-12 linolelaidic acid medium gave credence to this theory. We propose an amendment to the current scheme of the carbon flux through beta-oxidation taking into account the dispensability of beta-oxidation auxiliary enzymes for metabolizing trans double bonds at even-numbered positions.

Predicting the function and subcellular location of Caenorhabditis elegans proteins similar to Saccharomyces cerevisiae beta-oxidation enzymes.

The role of peroxisomal processes in the maintenance of neurons has not been thoroughly investigated. We propose using Caenorhabditis elegans as a model organism for studying the molecular basis underlying neurodegeneration in certain human peroxisomal disorders, e.g. Zellweger syndrome, since the nematode neural network is well characterized and relatively simple in function. Here we have identified C. elegans PEX-5 (C34C6.6) representing the receptor for peroxisomal targeting signal type 1 (PTS1), defective in patients with such disorders. PEX-5 interacted strongly in a two-hybrid assay with Gal4p-SKL, and a screen using PEX-5 identified interaction partners that were predominantly terminated with PTS1 or its variants. A list of C. elegans proteins with similarities to well-characterized yeast beta-oxidation enzymes was compiled by homology probing. The possible subcellular localization of these orthologues was predicted using an algorithm based on trafficking signals. Examining the C termini of selected nematode proteins for PTS1 function substantiated predictions made regarding the proteins' peroxisomal location. It is concluded that the eukaryotic PEX5-dependent route for importing PTS1-containing proteins into peroxisomes is conserved in nematodes. C. elegans might emerge as an attractive model system for studying the importance of peroxisomes and affiliated processes in neurodegeneration, and also for studying a beta-oxidation process that is potentially compartmentalized in both mitochondria and peroxisomes.

Reactive oxygen species as second messengers? Induction of the expression of yeast catalase T gene by heat and hyperosmotic stress does not require oxygen.

It is shown that oxygen is not absolutely needed for stress-induced synthesis of catalase T in the yeast Saccharomyces cerevisiae. Yeast cells develop heat resistance after exposure to elevated temperatures in anoxia. The levels of catalase activity and thermotolerance are comparable to those in aerobically stressed cells. While these results obviously do not exclude a stress signaling role of reactive oxygen species in some systems, as postulated by other authors, they suggest that the question of the obligatory requirement for reactive oxygen species in other stress signaling systems should be rigorously re-investigated.

Response of Saccharomyces cerevisiae to severe osmotic stress: evidence for a novel activation mechanism of the HOG MAP kinase pathway.

The HOG/p38 MAP kinase route is an important stress-activated signal transduction pathway that is well conserved among eukaryotes. Here, we describe a novel mechanism of activation of the HOG pathway in budding yeast. This mechanism operates upon severe osmostress conditions (1.4 M NaCl) and is independent of the Sln1p and Sho1p osmosensors. The alternative input feeds into the HOG pathway MAPKK Pbs2p and requires activation of Pbs2p by phosphorylation. We show that, upon severe osmotic shock, Hog1p nuclear accumulation and phosphorylation is delayed compared with mild stress. Moreover, both events lost their transient pattern, presumably because of the absence of negative feedback mediated by Ptp2p tyrosine phosphatase, which we found to be localized in the nucleus. Under severe osmotic stress conditions, the delayed nuclear accumulation correlates with a delay in stress-responsive gene expression. Severe osmoshock leads to a situation in which active and nuclear-localized Hog1p is transiently unable to induce transcription of osmotic stress-responsive genes. It also appeared from our studies that the Sho1p osmosensor is less active under severe osmotic stress conditions, whereas the Sln1p/Ypd1p/Ssk1p sensor and signal transducer functions normally under these circumstances.

Rpg1p, the subunit of the Saccharomyces cerevisiae eIF3 core complex, is a microtubule-interacting protein.

The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex. In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM). In budded cells, a portion of Rpg1p was obviously co-localised with microtubules. In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip. A punctate fluorescence pattern was typical for separated unbudded cells. Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p. In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed. No staining was observed in Rpg1p-depleted cells. In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules. We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae.

Nutritional control of nucleocytoplasmic localization of cAMP-dependent protein kinase catalytic and regulatory subunits in Saccharomyces cerevisiae.

In budding yeast, cAMP-dependent protein kinase (PKA) plays a central role in the nutritional control of metabolism, cell cycle, and transcription. This study shows that both the regulatory subunit Bcy1p and the catalytic subunit Tpk1p associated with it are predominantly localized in the nucleus of rapidly growing cells. Activation of nuclear PKA by cAMP leads to fast entry of a significant part of Tpk1p into the cytoplasm, while the regulatory subunit remains nuclear. In contrast to rapidly proliferating cells, both Bcy1p and Tpk1p are distributed over nucleus and cytoplasm in cells growing on a nonfermentable carbon source or in stationary phase cells. These results demonstrate that at least two different mechanisms determine the subcellular localization of PKA; cAMP controls the localization of Tpk1p, and the carbon source determines that of Bcy1p. The N-terminal domain of Bcy1p serves to target it properly during logarithmic and stationary phase. Studies with Bcy1p mutant versions unable to concentrate in the nucleus revealed that cells producing them are less viable in stationary phase than wild type cells, display delayed reproliferation following transfer to fresh growth medium, and, as diploids, exhibit reduced efficiency of sporulation.

Adr1p-dependent regulation of the oleic acid-inducible yeast gene SPS19 encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase.

The role of Saccharomyces cerevisiae Adr1p was examined with respect to the transcriptional regulation of the SPS19 gene encoding the peroxisomal beta-oxidation auxiliary enzyme 2,4-dienoyl-CoA reductase. The SPS19 promoter contains both an oleate response element that binds the Pip2p-Oaf1p transcription factor as well as a canonical Adr1p-binding element, termed UAS1(SPS19). Northern analysis demonstrated that transcriptional up-regulation of SPS19 was abolished in cells devoid of Adr1p. Expression of an SPS19-lacZ reporter gene was shown to be quiescent in the adr1Delta mutant and abnormally elevated in cells containing multiple ADR1 copies. UAS1(SPS19) was able to compete for formation of a specific complex between recombinant Adr1p-LacZ and UAS1(CTA1) representing the corresponding Adr1p-binding element in the promoter of the catalase A gene, and to interact directly with this fusion protein. We conclude that in the presence of fatty acids in the medium transcription of SPS19 is directly regulated by both Pip2p-Oaf1p and Adr1p.

Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta3-Delta2-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids.

Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids. However, their function during this process has not been demonstrated. Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment. The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p). A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid [cis-C(18:1(6))] medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu). We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal. Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid [cis-C(18:1(9))] medium irrespective of an SKL extension. Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes. Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes. The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.

A novel element in the promoter of the Saccharomyces cerevisiae gene SPS19 enhances ORE-dependent up-regulation in oleic acid and is essential for de-repression.

In Saccharomyces cerevisiae cells grown on oleic acid, genes encoding enzymes of beta-oxidation are induced by the interaction of a transcription factor composed of Pip2p and Oaflp with an oleate response element (ORE) in their promoters. The SPS19 gene, which encodes peroxisomal 2,4-dienoyl-CoA reductase, an auxiliary beta-oxidation enzyme, has been shown previously to be up-regulated by a canonical ORE. To determine whether additional elements contribute to this transcriptional upregulation, deletion analysis of the SPS19 promoter was conducted using SPS19-lacZ reporter genes. In a reporter construct containing a deletion adjacent to the ORE, transcriptional activation of SPS19 in oleic acid medium was impaired. Together with an additional segment that overlaps a portion of the canonical ORE, this region forms a continuous element (termed UAS(SPS19)) that is essential for de-repression of SPS19 when glucose levels are low. The potentially bi-partite UAS(SPS19) element was able to initiate bi-directional transcription from a promoterless CYC1-lacZ reporter construct under de-repression conditions, whereas the canonical ORE was not. In oleic acid-containing medium, UAS(SPS19) stimulated transcription of the reporter gene 2.4-fold compared to the intact SPS19 ORE, but did so only in the presence of Pip2p and Oaf1p. UAS(SPS19), which is similar to a transcriptional enhancer in the promoter of the sporulation-specific gene SPS4, was shown specifically to bind several proteins, including Pip2p and Oaflp. We propose that UAS(SPS19) and other sequences like it are required to enhance the transcriptional effects mediated by more specific response elements.

The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3.

The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.

Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae. Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase.

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein. Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.

Nucleocytoplasmic traffic of MAP kinases.

MAPK pathways represent a unique extracellular signal response system. An important feature of such a multicomponent system appears to be the spatial intracellular organization of individual components. Recent studies demonstrate that the MAP kinases of such pathways are the molecular link between the plasma membrane sensors and the nuclear transcription factors. Stimulation of several MAPK pathways induces rapid and transient nuclear accumulation of MAP kinases. Investigations on the mode of regulation of this process using higher eukaryotes Erk2 and lower eukaryotes Hog1 and Sty1/Spc1 have revealed that at least three events contribute to signal-induced nuclear localization of these MAP kinases: activation by phosphorylation, regulated nuclear import and export, and nuclear retention.