QSR1, an Essential Yeast Gene with a Genetic Relationship to a Subunit of the Mitochondrial Cytochromebc 1 Complex, Codes for a 60 S Ribosomal Subunit Protein
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.13372
ISSN1083-351X
AutoresFrederick A. Dick, Spyridoula Karamanou, Bernard L. Trumpower,
Tópico(s)DNA Repair Mechanisms
ResumoQSR1 (quinol-cytochromec reductase subunit-requiring) is a highly conserved, essential gene in Saccharomyces cerevisiae that was identified through a synthetic lethal screen by its genetic relationship to QCR6, the gene for subunit 6 (Qcr6p) of the mitochondrial cytochrome bc 1complex. The function of the QSR1-encoded protein (Qsr1p) and its relationship to the QCR6-encoded protein are unknown.When yeast cell lysates are fractionated by density gradient centrifugation, Qsr1p separates from organelles and sediments with a uniformly sized population of particles that are similar to eukaryotic ribosomes upon velocity gradient centrifugation. When 40 S and 60 S ribosomal subunits are separated on velocity gradients, Qsr1p is found exclusively with the 60 S subunits, where it is a stoichiometric component. Extracts prepared from qsr1-1 cells are defective in in vitro translation assays relative to the wild type.In yeast cell lysates in which QCR6 rescues an otherwise lethal qsr1-1 mutation, Qcr6p is found only in mitochondria, both in respiratory-competent cells and in rho0 cells in which the bc 1 complex is no longer present. These results suggest that suppression of theqsr1-1 mutation by QCR6 occurs by atrans-relationship across the outer mitochondrial membrane. QSR1 (quinol-cytochromec reductase subunit-requiring) is a highly conserved, essential gene in Saccharomyces cerevisiae that was identified through a synthetic lethal screen by its genetic relationship to QCR6, the gene for subunit 6 (Qcr6p) of the mitochondrial cytochrome bc 1complex. The function of the QSR1-encoded protein (Qsr1p) and its relationship to the QCR6-encoded protein are unknown. When yeast cell lysates are fractionated by density gradient centrifugation, Qsr1p separates from organelles and sediments with a uniformly sized population of particles that are similar to eukaryotic ribosomes upon velocity gradient centrifugation. When 40 S and 60 S ribosomal subunits are separated on velocity gradients, Qsr1p is found exclusively with the 60 S subunits, where it is a stoichiometric component. Extracts prepared from qsr1-1 cells are defective in in vitro translation assays relative to the wild type. In yeast cell lysates in which QCR6 rescues an otherwise lethal qsr1-1 mutation, Qcr6p is found only in mitochondria, both in respiratory-competent cells and in rho0 cells in which the bc 1 complex is no longer present. These results suggest that suppression of theqsr1-1 mutation by QCR6 occurs by atrans-relationship across the outer mitochondrial membrane. All cytochrome bc 1 complexes contain three redox proteins, cytochrome b, cytochromec 1, and an iron-sulfur protein, which are essential for the electron transfer and energy transduction functions of this enzyme in respiration and photosynthesis (1Trumpower B.L. J. Biol. Chem. 1990; 265: 11409-11412Abstract Full Text PDF PubMed Google Scholar). The cytochromebc 1 complexes of mitochondria also contain seven or eight additional subunits that lack prosthetic groups and that are not present in the bc 1 complexes of prokaryotes (2Trumpower B.L. Microbiol. Rev. 1990; 54: 101-129Crossref PubMed Google Scholar). The functions of these supernumerary subunits in the mitochondrial enzymes are largely unknown. QCR6 is the nuclear encoded gene for subunit 6 (Qcr6p) of the mitochondrial cytochrome bc 1 complex. Deletion of QCR6 does not impair growth of yeast on respiratory substrates at temperatures up to 35 °C, indicating that the supernumerary subunit 6 is not essential for respiration, although the deletion does result in a temperature-sensitive petite phenotype at 37 °C (3Yang M. Trumpower B.L. J. Biol. Chem. 1994; 269: 1270-1275Abstract Full Text PDF PubMed Google Scholar). To test whether the deletion of QCR6 might be covered by another, functionally redundant gene, we screened for mutants that require the nonessential subunit 6 and identifiedquinol-cytochrome c reductasesubunit-requiring mutants in two complementation groups, which we named qsr1 andqsr2 (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Surprisingly, the qsr mutants requireQCR6 to grow on either fermentable or nonfermentable carbon sources. This suggests that subunit 6 of the cytochromebc 1 complex has a role outside of respiration. QCR6 suppresses an otherwise lethal missense mutation in aqsr1-1 mutant. We cloned QSR1 by complementing the qsr1-1 mutant for growth in the absence ofQCR6 and showed that QSR1 is an essential gene in yeast (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The protein encoded by QSR1 is highly conserved and has been identified in at least 10 different eukaryotic organisms (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 5Dowdy S.F. Lai K.M. Weissman B.E. Matsui Y. Hogan B.L.M. Stanbridge E.J. Nucleic Acids Res. 1991; 19: 5763-5769Crossref PubMed Scopus (103) Google Scholar, 6Eisinger D.P. Jiang H.P. Serrero G. Biochem. Biophys. Res. 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Despite being identified in numerous organisms, little is known about the function of theQSR1-encoded protein. For the most part, the only information available has been the details of the molecular cloning of these genes, and the deduced amino acid sequences give few clues as to Qsr1p function. In addition to showing that QSR1 is essential, we showed that Qsr1p is localized throughout the cytoplasm in a punctate staining pattern (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In an effort to better understand the relationship between Qcr6p and Qsr1p, we have investigated the localization of these two proteins. Here we report that Qsr1p is a 60 S ribosomal subunit protein and that Qcr6p is found only inside mitochondria in yeast cells in which QCR6 rescues the qsr1-1 mutation. Yeast extract, peptone, Tryptone, and yeast nitrogen base without amino acids were purchased from Difco. Dextrose was obtained from Fisher. Yeast lytic enzyme was from ICN Biochemicals. Sucrose was from Life Technologies, Inc. Cycloheximide, heparin (sodium salt, M r 3000), protease inhibitors, and Nycodenz were purchased from Sigma. DNA-modifying enzymes were purchased from New England Biolabs Inc., Life Technologies, Inc., and Boehringer Mannheim. Oligonucleotides were purchased from DNA Express (Fort Collins, CO) and the Molecular Biology Core Facility at Dartmouth College. Wild-type cell lysates were prepared from Saccharomyces cerevisiae strains W303-1A (MATa) and W303-1B (MATα, ade2-1, his3-11,15, ura3-1, leu2-3,112, trp1-1, can1-100), which were obtained from Dr. R. Rothstein (Columbia University). The qsr1-1 mutant strain used in this study was LK10 (qsr1-1 with pLK57 instead of pMES32 (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar)), except in Figs. 6 and 7 b, where MMY3-3B was used (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The qcr6 deletion strain used was MES8 (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). rho0 strains were created from the relevant rho+ parents by treatment with ethidium bromide (11Goldring E.S. Grossman L.I. Krupnick D. Cryer D.R. Marmur J. J. Mol. Biol. 1970; 52: 323-335Crossref PubMed Scopus (351) Google Scholar) and genotyped on selective media. S. cerevisiae strains were grown in media prepared as described (12Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar), except that amino acid dropout mixtures were purchased from BIO 101, Inc. Plasmids were amplified in Escherichia coli strain DH5α (psi80dlacZΔM15, endA1,recA1, hsdR17(rk−mk−),supE44, thi-1, λ−, gyrA96, relA1, Δ(laczya-argF)U169, F−) (13Hanahan D. DNA Cloning. IRL Press, Oxford1985Google Scholar).Figure 7Qcr6p is localized exclusively within mitochondria. Affinity-purified antibodies were used to probe a Western blot of whole cell lysates prepared from wild-type (WT) and qcr6 deletion strains (a). Lysates from wild-type and qsr1-1 mutant strains were centrifuged at 16,000 × g to obtain a cytosolic supernatant (S) and a pellet containing mitochondria (P), which were then analyzed by Western blotting (b). Mitochondria from wild-type, qcr6 deletion, and qsr1-1 strains were treated with proteinase K before Western analysis (c).View Large Image Figure ViewerDownload Hi-res image Download (PPT) An 800-ml culture of W303-1B cells was grown in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) to a density of 0.6 absorbance units at 600 nm. Cells were harvested by centrifugation at 1500 × g for 5 min, resuspended in 5 ml of H2O containing 0.5% β-mercaptoethanol, and centrifuged as before. Pellets were resuspended in 20 ml of 1.2 m sorbitol and 50 mm KH2PO4, pH 7.5, containing 0.1% β-mercaptoethanol and transferred to a 250-ml flat-bottomed flask, and yeast lytic enzyme was added to a final concentration of 0.25 mg/ml. The cell suspension was placed on a shaker at 30 °C for 20–30 min. Spheroplast formation was monitored by checking for osmotic lysis of 1 μl of cells diluted in 10 μl of H2O (14Wise J.A. Methods Enzymol. 1991; 194: 405-415Crossref PubMed Scopus (56) Google Scholar). Spheroplasts were harvested by centrifugation at 2500 ×g for 1 min at 4 °C; resuspended in 15 ml of 25 mm HEPES, 5 mm magnesium acetate, and 500 mm sorbitol, pH 7.6; and centrifuged as before. Spheroplasts were then resuspended in 12 ml of 25 mm HEPES, 5 mm magnesium acetate, and 10% Nycodenz, pH 7.6, supplemented with 1 μg/ml pepstatin A, 10 μm leupeptin, 0.5 mm 1,10-phenanthroline, and 1 mmdiisopropyl fluorophosphate and transferred to a cold Dounce homogenizer. Spheroplasts were lysed on ice with 15 strokes of the loose plunger and 10 of the tight plunger. This resulted in ∼90% lysis as judged by viewing the lysate under a light microscope. The crude lysate was centrifuged at 2500 × g for 5 min to clear debris and unlysed cells. The supernatant was removed, and samples were saved for SDS-PAGE, 1The abbreviation used is: PAGE, polyacrylamide gel electrophoresis. protein determinations, and enzyme assays. The lysate cleared of debris and unlysed cells was centrifuged at 8000 × g for 15 min to sediment intact nuclei and plasma membranes. Samples of this medium-speed supernatant were taken for SDS-PAGE, protein determinations, and enzyme assays. Nycodenz density gradients were prepared by adding a 6-ml aliquot of HEND15 buffer (HEND15, HEND20, HEND25, HEND30, and HEND35 buffers contain 25 mm HEPES; 1 mm EDTA; 15, 20, 25, 30, and 35% Nycodenz, respectively; and 1 mm dithiothreitol, pH 7.6) to the bottom of a centrifuge tube. This buffer was then underlaid with HEND20 buffer, which was in turn underlaid with HEND25, HEND30, and HEND35 buffers. A 6-ml sample of the medium-speed supernatant was layered on top of this gradient and centrifuged at 120,000 × g in an SW 28 rotor for 10 h. The bottom of the centrifuge tube was punctured; 25 × 1.4-ml fractions were collected; and aliquots were taken for SDS-PAGE, protein determinations, refractometry, and enzyme assays (15Kolling R. Hollenberg C.P. EMBO J. 1994; 13: 3261-3271Crossref PubMed Scopus (271) Google Scholar, 16Rexach M.F. Latterich M. Schekman R.W. J. Cell Biol. 1994; 126: 1133-1148Crossref PubMed Scopus (94) Google Scholar). Fractions containing Qsr1p from the equilibrium density gradient were combined, and the Nycodenz concentration was adjusted to 35% by adding stock 80% Nycodenz. Two ml of this sample was used to underlay a gradient composed of 2 ml each of HEND30, HEND25, HEND20, and HEND15. This gradient was centrifuged at 180,000 × g in an SW 40 rotor for 16 h, after which 10 × 1-ml samples were collected, and aliquots were withdrawn for SDS-PAGE, protein determinations, refractometry, and enzyme assays (16Rexach M.F. Latterich M. Schekman R.W. J. Cell Biol. 1994; 126: 1133-1148Crossref PubMed Scopus (94) Google Scholar). The markers for soluble cytosolic proteins, mitochondria, vacuoles, and endoplasmic reticulum were Hxk1p/Hxk2p (both isoforms), Atp2p (F1β), Pho8p, and Sec61p, respectively. These markers were assayed by SDS-PAGE (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) and Western blotting (18Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). Qsr1p was detected by Western blotting with antiserum diluted 1:1000 (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). For quantitation, Western blots were repeated with three different protein dilutions to control for saturation of the nitrocellulose and visualized with the Lumiglo chemiluminescent system (Kirkegaard & Perry Laboratories, Inc.) at multiple time points to control for saturation of the band intensity on the film. Band intensities on blots were determined by scanning the film with an Apple Color One™ scanner, and the densities were quantified with the program NIH Image 1.52. Activity is defined as pixels, and relative activities were calculated by scaling the maximum activity for each marker to 100%. The GDPase marker for the Golgi apparatus was assayed enzymatically (19Abeijon C. Orlean P. Robbins P.W. Hirschberg C.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6935-6939Crossref PubMed Scopus (133) Google Scholar), and the maximum activity was scaled to 100%. Marker recoveries were calculated as activity per μl × volume divided by activity per μl of lysate × volume of lysate for each marker. Nycodenz concentrations were determined on an Abbe refractometer, and protein determinations were performed by the Bio-Rad method according to the manufacturer's instructions. Velocity gradient centrifugations of lysates were done essentially as described by Ramirez et al. (20Ramirez M. Wek R. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (123) Google Scholar). Lysates of W303-1B cells were prepared in 20 mm Tris, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol, 0.2 mg/ml heparin, 1 μg/ml pepstatin A, 10 μm leupeptin, and 1 mm diisopropyl fluorophosphate. The lysate was then centrifuged at 15,000 ×g for 30 min to remove debris and most organelles. Supernatant equivalent to 35 absorbance units at 600 nm was loaded onto 5–47% sucrose gradients prepared in 20 mm Tris, pH 7.5, 50 mm NaCl, 1 mm dithiothreitol, 1 μg/ml pepstatin A, and 10 μm leupeptin. The gradients were then centrifuged at 220,000 × g for 3 h and 45 min in an SW 40 rotor and were collected in 20 × 0.5-ml fractions. Absorbance was monitored at 254 nm. Yeast lysates and gradient buffers were supplemented with 50 μg/ml cycloheximide, 10 mmMgCl2, or 0.5 m KCl where indicated. Samples were analyzed by SDS-PAGE and Western blotting as described above. Blots were probed with rabbit polyclonal antibodies to Qsr1p or mouse monoclonal antibodies to the ribosomal L3 protein (diluted 1:2500). The relative amounts of Qsr1p and L3 associated with 60 S subunits, 80 S ribosomes, and polysomes were determined by scanning Western blots of fractions from a gradient of polysomes from yeast cells in which protein synthesis was arrested by addition of cycloheximide. Band intensities of the two proteins were quantified with the NIH Image program as described above. Ribosomes were isolated as described by Raue et al. (21Raue H.A. Mager W.H. Planta R.J. Methods Enzymol. 1991; 194: 453-477Crossref PubMed Scopus (28) Google Scholar). 60 S subunits equivalent to 2 absorbance units at 260 nm were treated with 20 μg/ml trypsin for 0, 5, 15, and 30 min, either in the absence or presence of equimolar amounts of 40 S subunits as indicated. Reactions were stopped by adding SDS-PAGE sample buffer preheated to 95 °C. Immunofluorescence microscopy was performed on W303-1B cells as described previously (8Marty I. Brugidou C. Chartier Y. Meyer Y. Plant J. 1993; 4: 265-278Crossref PubMed Scopus (56) Google Scholar, 20Ramirez M. Wek R. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (123) Google Scholar). Antibodies to the L3 protein were used at a dilution of 1:200. S100 extracts were prepared from wild-type or qsr1-1 mutant cells (23Deshaies R.J. Schekman R. J. Cell Biol. 1989; 109: 2653-2664Crossref PubMed Scopus (133) Google Scholar) and stored at a concentration of 10 mg/ml. Thawed extracts were treated with nuclease, and reactions were carried out at 20 °C at a final protein concentration of 6 mg/ml in the reaction buffer described by Leibowitzet al. (24Leibowitz M.J. Barbone F.P. Georgopoulos D.E. Methods Enzymol. 1991; 194: 536-545Crossref PubMed Scopus (12) Google Scholar). Total yeast RNA was prepared from spheroplasts using the RNeasy midikit from QIAGEN Inc., and poly(A)+ RNA was purified on oligo(dT)-cellulose (12Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar). Reactions were stopped by spotting on trichloroacetic acid-soaked filter paper. Filters were washed extensively with 10% trichloroacetic acid and ethanol and dried, and incorporation of [35S]methionine into trichloroacetic acid-insoluble material was quantitated by liquid scintillation. DNA manipulations were carried out according to the manufacturers' directions and standard procedures (11Goldring E.S. Grossman L.I. Krupnick D. Cryer D.R. Marmur J. J. Mol. Biol. 1970; 52: 323-335Crossref PubMed Scopus (351) Google Scholar). DNA was sequenced with the Prism dye terminator cycle sequencing kit from Applied Biosystems Inc. (ABI) or the modified FS kit. Reactions were analyzed on an ABI 373 automated sequencer at the Dartmouth Molecular Biology Core Facility, and the data were interpreted using Sequence Editor™ (ABI). The plasmid pLK57 was obtained from the original library screening to complement the qsr1-1 mutant (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). It was identified as aQCR6-containing suppressor because it contains theQCR6 genomic 1.9-kilobase SphI fragment. pLK13, which expresses the glutathione S-transferase-QCR6p fusion, was constructed by amplifying the QCR6 open reading frame by polymerase chain reaction. The polymerase chain reaction mixtures were initially denatured at 95 °C for 3 min and then cycled 25 times at 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 2 min, and the resulting product was subcloned into the EcoRI sites of pGEX-1λT. The primers used were 1QCR6 (5′-GAA TTC ATG TTG GAA CTA GTT GGT GAG-3′) and 2QCR6 (5′-GAA TTC CTA CTT TAA TTT GTC AAA TAA TCT AGG-3′). Antibodies to a glutathioneS-transferase-Qcr6p fusion protein were raised in rabbits (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), affinity-purified (22Pringle J.R. Adams A.E.M. Drubin D.G. Haarer B.K. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (601) Google Scholar), and used at a dilution of 1:500. Lysates of wild-type andqsr1-1 mutant strains were prepared by resuspending cells from a 5-ml culture in 50 mm Tris-Cl, pH 7.4, 400 mm mannitol, and 2 mm EDTA and vortexing with glass beads. Mitochondria were then pelleted by centrifugation at 16,000 × g for 15 min in a microcentrifuge. Samples for SDS-PAGE were prepared from the supernatant (cytosol) and the pellet (mitochondria). Mitochondria with bound polysomes were purified with the buffers adjusted to pH 6.0 to minimize contamination by microsomes (25Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (305) Google Scholar, 26Ades I.Z. Butow R.A. J. Biol. Chem. 1980; 255: 9918-9924Abstract Full Text PDF PubMed Google Scholar). The pellet fraction containing mitochondria with bound polysomes was loaded onto a 30–80% sucrose step gradient in buffer containing 200 μg/ml cycloheximide and centrifuged at 120,000 × gfor 16 h, and gradients were fractionated as described above. Mitochondria were treated with proteinase K as described by Hartlet al. (27Hartl F.-U. Schmidt B. Wachter E. Weiss H. Neupert W. Cell. 1986; 47: 939-957Abstract Full Text PDF PubMed Scopus (199) Google Scholar). The punctate cytoplasmic localization of Qsr1p (4Tron T. Yang M. Dick F.A. Schmitt M.E. Trumpower B.L. J. Biol. Chem. 1995; 270: 9961-9970Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) implies that it is concentrated in some subcellular compartment or is oligomerized to create regions of high density when viewed by immunofluorescence microscopy. When lysates from exponentially grown yeast cells were centrifuged to remove nuclei, plasma membranes, and larger organelles, 95% of the Qsr1p was recovered in the supernatant (TableI). When this medium-speed supernatant was fractionated on a Nycodenz gradient, Qsr1p sedimented as a uniform population as shown in Fig. 1. The distribution of Qsr1p overlaps with, but does not exactly match that of Sec61p, an integral membrane protein that is part of the protein translocation complex of the endoplasmic reticulum.Table IRecovery of Qsr1p and organelle markers after fractionation of yeast cell lysatesMarkerOrganelle/compartmentMarker recoveryLysateMSS1-aMSS, medium-speed supernatant (described under “Experimental Procedures”).Pool 1Pool 2%Qsr1p100958583Sec61pEndoplasmic reticulum1002660Atp2pMitochondria1003452HxkpCytosol1009500GDPaseGolgi1009500Pho8pVacuole1005540Recoveries were calculated by dividing the activity per μl × volume for each marker by the activity per μl × volume of the lysate. The percent recovery of each marker in the lysate is scaled to 100 for comparison. Pool 1 is fractions 11–15 from the first Nycodenz gradient (Fig. 1), and Pool 2 is the bottom two fractions from the flotation gradient described under “Experimental Procedures.”1-a MSS, medium-speed supernatant (described under “Experimental Procedures”). Open table in a new tab Recoveries were calculated by dividing the activity per μl × volume for each marker by the activity per μl × volume of the lysate. The percent recovery of each marker in the lysate is scaled to 100 for comparison. Pool 1 is fractions 11–15 from the first Nycodenz gradient (Fig. 1), and Pool 2 is the bottom two fractions from the flotation gradient described under “Experimental Procedures.” To investigate the possibility that Qsr1p is associated with a subpopulation of endoplasmic reticulum, the fractions containing Qsr1p from the first Nycodenz gradient were combined and applied to the bottom of a flotation gradient. In this procedure, membranes floated up through the gradient until they came to equilibrium, while denser proteins and protein complexes remained on the bottom. Under these conditions, Qsr1p remained at the bottom of the gradient while Sec61p floated up (data not shown). Qsr1p was thus separated completely from all markers, except for 2% of the Atp2p mitochondrial marker, which was recovered at the bottom of the second gradient (Table I). That Qsr1p did not float in the second gradient indicates that it is not associated with any membranes. During the 10-h centrifugation of the first density gradient, membranes will reach their equilibrium density, but proteins and protein complexes will still be sedimenting toward their equilibrium position at the bottom of the tube and will be recovered according to their sedimentation rate rather than density. Since Qsr1p is predicted from the deduced amino acid sequence to be ∼25 kDa and the cytosolic markers Hxk1p and Hxk2p are 100-kDa dimers in solution (28Barman T.E. Enzyme Handbook. Springer-Verlag New York Inc., New York1969: 377-378Google Scholar), Qsr1p appears to be part of a large complex that sediments significantly faster than Hxk1p/Hxk2p in the first density gradient (Fig. 1). Quantitation of the amount of Qsr1p relative to that in the initial lysate (Table I) indicates that >80% of the Qsr1p was recovered after the second density gradient centrifugation as a large membrane-free complex under the standard conditions used to lyse and fractionate yeast cells. The size of the putative particle identified by the density gradient centrifugations was investigated by velocity gradient centrifugation and determined to be between 80 and 100 S under conditions that maintain association of ribosomal subunits (data not shown). This size estimate led us to investigate whether Qsr1p is associated with ribosomes. Rapidly sedimenting organelles and membranes were removed from yeast cell lysates by centrifugation for 30 min at 15,000 ×g (20Ramirez M. Wek R. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (123) Google Scholar), after which polysomes were separated by gradient centrifugation. The distribution of 40 and 60 S subunits, 80 S ribosomes, and polysomes was monitored by absorbance at 254 nm, and the distribution of Qsr1p and L3, a large ribosomal subunit protein (29Woolford Jr., J.L. Adv. Genet. 1991; 29: 63-118Crossref PubMed Scopus (63) Google Scholar), was monitored by Western analysis. The association of large and small ribosomal subunits is dependent upon the presence of Mg2+, and cycloheximide arrests ribosomes during translation on an mRNA template (20Ramirez M. Wek R. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (123) Google Scholar). We thus varied these parameters to test the association of Qsr1p with ribosomes. As shown in Fig. 2 a, when protein synthesis was inhibited with cycloheximide and Mg2+ was included, there were small amounts of 40 S and 60 S subunits followed by larger amounts of 80 S ribosomes and polysomes. L3 was localized to the 60 S and 80 S peaks and with the polysomes, as expected. A small amount of Qsr1p was with the 60 S subunit, and its distribution in 80 S and polysome fractions matched that of L3. When Mg2+ was omitted from the buffers and cycloheximide was included, 40 S and 60 S subunits dissociated, and no polysomes were detected as shown in Fig. 2 b. Under these conditions, L3 marked the 60 S subunit, and the distribution of Qsr1p matched that of L3, indicating that it is also a 60 S subunit protein. The experiment depicted in Fig. 2 c was performed to determine whether Qsr1p associates with the 40 S subunit prior to initiation of translation. With the omission of cycloheximide and inclusion of Mg2+, the ribosomes were able to finish translating in lysates, and translation initiation factors like Gcn2p are associated with the 40 S subunit (20Ramirez M. Wek R. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (123) Google Scholar). Under these conditions, Qsr1p was found with the 60 S subunit, 80 S ribosomes, and polysomes with the same distribution as the large ribosomal subunit marker L3. Quantitative Western blots indicate that Qsr1p constitutes between 0.1 and 0.5% of total cell protein (data not shown), which is in agreement with the abundance of other ribosomal proteins (29Woolford Jr., J.L. Adv. Genet. 1991; 29: 63-118Crossref PubMed Scopus (63) Google Scholar). Although Qsr1p was consistently found in 60 S subunits, visual comparison of the relative amounts of L3 and Qsr1p in the 60 S subunits and 80 S ribosomes in Fig.2 (a and c) indicated that proportionately less Qsr1p than L3 was present in free 60 S subunits. To investigate the stoichiometry of Qsr1p on 60 S subunits, the amounts of Qsr1p and L3 were quantitated by Western analysis of fractions from a gradient identical to that in Fig. 2 a. As shown in Fig.2 d, the ratio of Qsr1p to L3 was relatively constant and was close to 1 in fractions containing 80 S ribosomes and polysomes. In fractions 10 and 11, which included the 60 S subunits, Qsr1p was diminished ∼5-fold relative to L3. This suggests that although Qsr1p is quantitatively recovered with the ribosomal fraction (Fig. 1 and Table I), a portion of free 60 S subunits lack Qsr1p. To further characterize the association of Qsr1p with the large ribosomal subun
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