Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation

Mammalian and fungal prions arise de novo; however, the mechanism is poorly understood in molecular terms. One strong possibility is that oxidative damage to the non-prion form of a protein may be an important trigger influencing the formation of its heritable prion conformation. We have examined the oxidative stress-induced formation of the yeast [PSI+] prion, which is the altered conformation of the Sup35 translation termination factor. We used tandem affinity purification (TAP) and mass spectrometry to identify the proteins which associate with Sup35 in a tsa1 tsa2 antioxidant mutant to address the mechanism by which Sup35 forms the [PSI+] prion during oxidative stress conditions. This analysis identified several components of the cortical actin cytoskeleton including the Abp1 actin nucleation promoting factor, and we show that deletion of the ABP1 gene abrogates oxidant-induced [PSI+] prion formation. The frequency of spontaneous [PSI+] prion formation can be increased by overexpression of Sup35 since the excess Sup35 increases the probability of forming prion seeds. In contrast to oxidant-induced [PSI+] prion formation, overexpression-induced [PSI+] prion formation was only modestly affected in an abp1 mutant. Furthermore, treating yeast cells with latrunculin A to disrupt the formation of actin cables and patches abrogated oxidant-induced, but not overexpression-induced [PSI+] prion formation, suggesting a mechanistic difference in prion formation. [PIN+], the prion form of Rnq1, localizes to the IPOD (insoluble protein deposit) and is thought to influence the aggregation of other proteins. We show Sup35 becomes oxidized and aggregates during oxidative stress conditions, but does not co-localize with Rnq1 in an abp1 mutant which may account for the reduced frequency of [PSI+] prion formation

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the repiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency

Integrity of SRP RNA is ensured by La and the nuclear RNA quality control machinery

The RNA component of signal recognition particle (SRP) is transcribed by RNA polymerase III, and most steps in SRP biogenesis occur in the nucleolus. Here, we examine processing and quality control of the yeast SRP RNA (scR1). In common with other pol III transcripts, scR1 terminates in a U-tract, and ma-ture scR1 retains a U4–5 sequence at its 3 ′ end. In cells lacking the exonuclease Rex1, scR1 terminates in a longer U5–6 tail that presumably represents the primary transcript. The 3 ′ U-tract of scR1 is protected from aberrant processing by the La homologue, Lhp1 and overexpressed Lhp1 apparently competes with both the RNA surveillance system and SRP assem-bly factors. Unexpectedly, the TRAMP and exosome nuclear RNA surveillance complexes are also impli-cated in protecting the 3 ′ end of scR1, which accu-mulates in the nucleolus of cells lacking the activities of these complexes. Misassembled scR1 has a pri-mary degradation pathway in which Rrp6 acts early, followed by TRAMP-stimulated exonuclease degra-dation by the exosome. We conclude that the RNA surveillance machinery has key roles in both SRP biogenesis and quality control of the RNA, poten-tially facilitating the decision between these alterna-tive fates

Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd(+) bacterium as the result of replication errors or recombination-dependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with ≈600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage

GODLY PLAY SUOMESSA : Kyselytutkimus Godly play -uskontokasvatusmenetelmän käyttäjille

Hakulinen, Heidi. Godly play Suomessa – Kyselytutkimus Godly play -uskontokasvatusmenetelmän käyttäjille. Diak Etelä, Järvenpää, Kevät 2011, 54s., 1 liite. Diakonia-ammattikorkeakoulu, Sosiaalialan koulutusohjelma, Kristillisen lapsi- ja nuorisotyön suuntautumisvaihtoehto, sosionomi (AMK) + kirkon nuorisotyönohjaajan virkakelpoisuus + lastentarhanopettajan kelpoisuus. Tämän opinnäytetyön tarkoitus oli selvittää millaista Godly play -toimintaa Suomessa on ja kuinka paljon menetelmää käytetään. Vastaajat tavoitettiin Seurakuntaopiston kautta, joten kysely ei tavoittanut kaikkia Suomen Godly playn käyttäjiä. Kyselylomakkeita lähetettiin 34 käyttäjälle ja vastauksia saatiin 31. Tutkimus toteutettiin kvantitatiivisesti ja tutkimusaineisto kerättiin syksyllä 2010 sähköpostitse. Aineisto analysoitiin SPSS -ohjelmalla sekä sisällön analyysi -menetelmällä talvella 2010–2011. Tutkimuksessa selvisi, että Suomessa on vielä melko vähän Godly playn käyttäjiä. Menetelmää käytetään seurakunnan monilla eri työaloilla, mutta myös seurakunnan ulkopuolella. Eniten menetelmää käytetään seurakunnan päiväkerhossa, rippikoulussa sekä aikuisille suunnatuissa hartaushetkissä. Godly playtä käytetään vain osittain sen alkuperäisessä muodossaan. Reagointityöskentelyä käytetään yllättävän vähän siihen nähden, että sen sanotaan olevan koko menetelmän tärkein osa.Hakulinen, Heidi. Godly play in Finland – a Survey for the users of Godly play religious education method. 54p,. 1 appendix. Language: Finnish. Järvenpää, Spring 2011. Diaconia University of Applied Sciences. Degree Program in Social Services, Option in Christian Youth Work. Degree: Bachelor of Social Services. The purpose of the thesis was to find out what kind of Godly play activities there are in Finland and how often this religious education method is used. The respondents were contacted through the Church Training College, so that survey didn’t reach every Godly play user in Finland. The questionnaire was sent to altogether 34 users and responses were received 31. The research was carried out quantitatively and the data were collected in autumn 2010 via e-mail. The data were analyzed using the SPSS program and content analysis method in winter 2010–2011. The result showed that there are quite few Godly play users in Finland. The method is used most in the church day care, confirmation camps and hours of devotion for adults. In addition, it is used for example in day care centres and schools. Godly play is only partly used as it originally is supposed to be used. The session includes the response time surprisingly rarely taking into account that it is considered the most important part in the whole method. Keywords: Godly play, Montessori Method, Christian education, quantitative researc

Site-Specific Release of Nascent Chains from Ribosomes at a Sense Codon ▿

“2A” oligopeptides are autonomous elements containing a D(V/I)EXNPGP motif at the C terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyltransferase center. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation-terminating release factors eRF1 and eRF3 play key roles in the reaction. On the depletion of eRF1, a greater proportion of ribosomes extend through the 2A coding sequence, yielding the full-length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A peptide-containing protein inhibits the growth of cells compromised for release factor activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific “termination” reaction, despite the presence of a proline codon in the A site. After this, the majority of ribosomes continue translation, generating the separate downstream product

2A peptides provide distinct solutions to driving stop-carry on translational recoding

Funded by the U.K. Biotechnology and Biological Sciences Research Council (BB/E/01070911)Expression of viral proteins frequently includes non-canonical decoding events (‘recoding’) during translation. ‘2A’ oligopeptides drive one such event, termed ‘stop-carry on’ recoding. Nascent 2A peptides interact with the ribosomal exit tunnel to dictate an unusual stop codon-independent termination of translation at the final Pro codon of 2A. Subsequently, translation ‘reinitiates’ on the same codon, two individual proteins being generated from one open reading frame. Many 2A peptides have been identified, and they have a conserved C-terminal motif. Little similarity is present in the N-terminal portions of these peptides, which might suggest that these amino acids are not important in the 2A reaction. However, mutagenesis indicates that identity of the amino acid at nearly all positions of a single 2A peptide is important for activity. Each 2A may then represent a specific solution for positioning the conserved C-terminus within the peptidyl-transferase centre to promote recoding. Nascent 2A peptide:ribosome interactions are suggested to alter ribosomal fine structure to discriminate against prolyl-tRNAPro and promote termination in the absence of a stop codon. Such structural modifications may account for our observation that replacement of the final Pro codon of 2A with any stop codon both stalls ribosome processivity and inhibits nascent chain release.Publisher PDFPeer reviewe

Analysis of protein oxidation and aggregation in an <i>abp1</i> mutant.

<p><b>A.</b> Mutants disrupting the cortical actin cytoskeleton dot not affect Sup35 protein oxidative damage. The wild-type, <i>abp1</i>, <i>crn1</i> and <i>pan1</i> mutant strains were treated with 100μM hydrogen peroxide for 20 hours and protein carbonylation used as a measure of protein oxidative damage. Protein extracts were treated with the carbonyl-specific probe, DNPH, and analyzed by Western blot analysis using an antibody against DNPH. <b>B.</b> Subcellular distribution of Sup35 in wild-type and <i>abp1</i> mutant cells grown in the presence or absence of 100μM hydrogen peroxide for 20 hours. Total denotes total crude extract; Soluble, soluble fraction; Pellet, SDS-resistant high molecular weight fraction. <b>C.</b> Representative fluorescent micrographs are shown for wild-type and <i>abp1</i> mutant cells expressing GFP–Ubc9^ts. Strains were grown in SRaf media before switching to SGal media to induce GFP–Ubc9^ts expression for 3 hours. This was followed by a 37°C heat shock for 30 minutes to trigger the misfolding and aggregation of Ubc9. Quantification of the frequency of GFP–Ubc9 foci is expressed as the percentage of cells containing fluorescence foci out of approximately 300 cells counted. Error bars denote standard deviation.</p