Most methods for isolation of RNA from yeast require tedious vortexing with glass beads, and give low yields when scaled down to 10 ml cultures (1). In addition, it is frequently desirable to prepare RNA from several different yeast strains grown under a variety of growth conditions, and preparations using glass beads are impractical when dealing with multiple samples.Heating and freezing of yeast cells in the presence of phenol and SDS has been used for large scale isolation of RNA from yeast (2). We report here a mini-prep version of yeast RNA isolation using phenol and SDS that allows for the processing of a dozen samples in about 60 minutes, and provides enough RNA to run several northern blots. In our hands methods using glass beads are slower and give lower yields of RNA.RNA was isolated as follows. Ten ml cultures were grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD6W of 2.5-5.0. We have also used YP 2% galactose, YP 4% ethanol/3 % glycerol, YP 2% raffinose and a variety of minimal media. The cells were harvested by centrifugation and resuspended in 400 td of 50 mM Na acetate pH 5.3, 10 mM EDTA ('AE buffer').The resuspended cells were transferred to a 1.5 ml microcentrifuge tube and 40 IAI of 10% SDS was added. The suspension was vortexed and an equal volume of fresh phenol, previously equilibrated with AE buffer, was added. The mixture was again vortexed and incubated at 65°C for 4 min. The mixture was then rapidly chilled in a dry ice/ethanol bath until phenol crystals appeared, and then centrifuged for 2 min at maximum speed in a microcentrifuge to separate the aqueous and phenol phases. The upper, aqueous phase was transferred to a fresh microcentrifuge tube and extracted with phenol/chloroform at room temperature for 5 min. The extracted aqueous phase was then brought to 0.3 M Na acetate, pH 5.3, by adding 40 tl of 3 M Na acetate pH 5.3, after which 2.5 volumes of ethanol were added to precipitate the RNA. After washing with 80% ethanol, the pellet was dried and resuspended in 20 i1 of sterile water and stored at -70°C until used. Throughout the preparation, normal precautions to avoid ribonuclease contamination were taken (3).The isolation procedure was performed using seven different wild type yeast strains and over two dozen derivatives of these strains. Yields varied between 60 ,ag and 300 Mg of RNA per 10 ml culture, with an average yield of 135 ,ug as quantitated by absorbance at 260 nm. Fig. la shows the quality of the RNA that is isolated using this method. No degradation of ribisomal RNA bands is seen, and recoveries of both high and low molecular weight RNA appear excellent.

No abstract

No abstract

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.Most steps in ribosome synthesis take place in the nucleolus, a specialized subnuclear region. This process starts with the synthesis of two pre-rRNA transcripts, 35S and pre-5S rRNA, which are processed and base modified to yield the mature 25S/28S, 18S, 5.8S, and 5S rRNAs, respectively (18). During these processes about 80 ribosomal proteins assemble onto the rRNAs to yield preribosomal particles, which are exported into the cytoplasm (41). In contrast to pre-rRNA processing and modification, very little is known about the assembly pathway for eukaryotic ribosomal subunits or the features that make them competent for nuclear exit (for recent reviews, see references 18 and 40).The transport of macromolecules through the nuclear pores is thought to involve facilitated diffusion of soluble transport factors over the repeat sequences of the nuclear pore proteins (nucleoporins) that form and line the nuclear pore complex. Directionality of transport is provided by the small GTPase Ran, due to the presence of a step RanGTP/RanGDP gradient across the nuclear membrane (for a review, see reference 23). RanGTP binds with high affinity to nuclear import and export receptors (importins and exportins, respectively) of the karyopherin ␤ superfamily (10). For nuclear exit, export cargoes, which harbor nuclear export sequences (NESs) (e.g., leucinerich NESs), form an intranuclear complex with the NES receptor Xpo1p/Crm1 in the presence of RanGTP (8,33). This trimeric complex is then exported from the nucleus into the cytoplasm.Saccharomyces cerevisiae has been a useful system for the analysis of the nuclear pore complex as well as transport factors (6). We have reported an in vivo assay for ribosomal export in yeast that uses a fusion between green fluorescent protein (GFP) and ribosomal protein Rpl25p (15). Rpl25p is imported into the nucleus and assembles with ribosomes by direct binding to the rRNA inside the nucleolus (39). Passage of both the free Rpl25p-GFP and the preribosomal particles through the nucleoplasm appears to be rapid in wild-type cells, and GFP-labeled ribosomes were detected by fluorescence microscopy in the cytoplasm. Mutations causing defects in subunit e...

The cytochrome bc1 complex is an oligomeric electron transfer enzyme located in the inner membrane of mitochondria and the plasma membrane of bacteria. The cytochrome bc1 complex participates in respiration in eukaryotic cells and also participates in respiration, cyclic photosynthetic electron transfer, denitrification, and nitrogen fixation in a phylogenetically diverse collection of bacteria. In all of these organisms, the cytochrome bc1 complex transfers electrons from ubiquinol to cytochrome c and links this electron transfer to translocation of protons across the membrane in which it resides, thus converting the available free energy of the oxidation-reduction reaction into an electrochemical proton gradient. The mechanism by which the cytochrome bc1 complex achieves this energy transduction is the protonmotive Q cycle. The Q cycle mechanism has been documented by extensive experimentation, and recent investigations have focused on structural features of the three redox subunits of the bc1 complex essential to the protonmotive and electrogenic activities of this membranous enzyme.