Scp160p, an RNA-binding, Polysome-associated Protein, Localizes to the Endoplasmic Reticulum of Saccharomyces cerevisiae in a Microtubule-dependent Manner

• DOI: 10.1074/jbc.m009430200
• Publication Date: 2002-07-26T15:08:15Z
• Authors: Steffen Frey; Martin Pool; Matthias Seedorf
• Keywords: Polysome; Heterogeneous nuclear ribonucleoprotein

Scp160p is an RNA-binding protein containing 14 tandemly repeated heterogenous nuclear ribonucleoprotein K-homology domains, which are implicated in RNA binding. interacts with free and membrane-bound polysomes that dependent upon the presence of mRNA. Despite its on cytosolic polysomes, predominantly localized to endoplasmic reticulum (ER). Accumulation Scp160p-ribosome complexes at ER requires function microtubules but independent actin cytoskeleton. We propose multi-K-homology-domain functions as binding platform, interacting transported ER. RNP green fluorescent low salt polymerase chain reaction phosphate-buffered saline localization spatial restriction translation important mechanisms biological processes (reviewed Refs. 1Lipshitz H.D. Smibert C.A. Curr. Opin. Genet. Dev. 2000; 10: 476-488Crossref PubMed Scopus (143) Google Scholar and2Bashirullah A. Cooperstock R.L. Lipshitz Annu. Rev. Biochem. 1998; 67: 335-394Crossref (294) Scholar). The process mRNA initiated by association one or more proteins through a targeting signal most commonly located 3′-untranslated region transcripts. This may occur nucleus soon after transcription, resulting formation particles (3Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Bioichem. 1993; 62: 289-321Crossref (1369) Scholar), then exported into cytosol (4Krecic A.M. Swanson M.S. Cell Biol. 1999; 11: 363-371Crossref (722) Alternatively, such form cytosol. migrate along cytoskeleton their final destinations, where they anchored translated Ref. 5Jansen R.P. FASEB J. 13: 455-466Crossref (140) multistep depends specific trans-acting proteins. One common feature these factors repeats different domains (6Burd Dreyfuss Science. 1994; 265: 615-621Crossref (1752) These include members double-stranded family (7St Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. 1992; 89: 10979-10983Crossref (491) Scholar, 8Ferrandon Elphick L. Nusslein-Volhard C. St Cell. 79: 1221-1232Abstract Full Text PDF (356) homologues zipcode-binding (9Havin Git Elisha Z. Oberman F. Yaniv K. Schwartz S.P. Standart N. Yisraeli J.K. Genes 12: 1593-1598Crossref (183) 10Ross A.F. Oleynikov Y. Kislauskis E.H. Taneja K.L. Singer R.H. Mol. 1997; 17: 2158-2165Crossref RNP1 (11Carson J.H. Kwon Barbarese E. Neurobiol. 8: 607-612Crossref (117) 12Hoek K.S. Kidd G.J. Carson Smith R. Biochemistry. 37: 7021-7029Crossref (194) 13Cote Gautreau Denegre J.M. Kress T.L. Terry N.A. Mowry 4: 431-437Abstract (205) motifs involved (KH) domain, originally described RNP-K (14Siomi H. Michael W.M. Nucleic Acids Res. 21: 1193-1198Crossref (463) A KH domain consists ∼70 amino acids includes conserved hydrophobic core, invariant GXXG motif, additional variable segment. NMR structural studies individual revealed βααββα fold (15Musco Stier Joseph Castiglione Morelli M.A. Nilges Gibson T.J. Pastore 1996; 85: 237-245Abstract (252) 16Baber J.L. Libutti Levens Tjandra 289: 949-962Crossref (87) In yeast, Scp160p, copies has been identified (17Wintersberger Kuhne Karwan Yeast. 1995; 929-944Crossref (68) localizes enrichment scp160Δ mutants viable, display defects cell morphology segregation, cells increased size DNA content per Scholar,18Jackson C.L. Kepes Genetics. 137: 423-437Crossref mechanism this complex phenotype established Δscp160 unknown. Drosophila, functional homologue also identified. protein, DDP1, binds dodeca satellite repeat regions centromeric heterochromatin embryonic larval nuclei (19Cortes Huertas Fanti Pimpinelli Marsellach F.X. Pina B. Azorin EMBO 18: 3820-3833Crossref (66) 20Cortes 20: 3860-3869Crossref (38) Overexpression DDP1 complements segregation Similar multi KH-domain found ubiquitously all eukaryotic cells. vertebrate species known vigilin (21Schmidt Henkel Poschl Zorbas Purschke W.G. Gloe T.R. Muller P.K. Eur. 206: 625-634Crossref (57) 22Plenz Gan Raabe H.M. Tissue 273: 381-389Crossref (28) 23Dodson R.E. Shapiro D.J. Chem. 272: 12249-12252Abstract (91) clear picture cellular targets not yet emerged. Northwestern blot analyses demonstrated activity vitro sequence specificity (24Weber V. Wernitznig Hager Harata Frank P. Wintersberger 249: 309-317Crossref (34) gel mobility shift assays affinity column chromatography showed Xenopus specifically vitellogenin (23Dodson 25Kanamori Dodson 3991-4003Crossref (49) stabilizing blocking cleavage endonuclease (26Cunningham Nagel Schoenberg D.R. 97: 12498-12502Crossref (76) contrast results, Kruse et al. (27Kruse Grunweller Willkomm D.K. Pfeiffer T. Hartmann R.K. 329: 28Kruse Vollbrandt Sommer Busch Brinkmann 346: 107-115Crossref (41) Scholar) human be transport tRNA. Recently, was reported associated component messenger poly(A)-binding released EDTA treatment (29Lang B.D. Fridovich-Keil 28: 1576-1584Crossref (48) observations contradictory previously membrane To resolve apparent discrepancy, we decided investigate intracellular distribution carefully ask whether both polysomes. could show enriched mRNA-dependent manner. Interestingly, demonstrate intact microtubules. replace SCP160 engineered gene encoding Scp160-GFP, amplified PCR fragment from +2465 stop codon using 5′-CACCGATCCAAAGGCTCA-3′ 5′-CCGctcgagATCTTCTTAAGGATTTCAAAACC-3′ primers genomic (YEP13/6 Scholar)) template. cloned first TOPOII vector (TOPO TA cloning kit, Invitrogen), re-excised SacI andXhoI, fused in-frame mutant (S65T, V163A) GFP NUF2 non-replicating plasmid pPS1539 (30Seedorf Damelin Kahana Taura Silver P.A. 19: 1547-1557Crossref (118) plasmid, pMS356, linearized Tth111I used for transformation S288c-derived diploid yeast strain. Ura+ transformants were selected checked fluorescence microscopy expression Scp160p-GFP. confirm replacement, strain sporulated, Scp160p-GFP-expressing spores compared wild-type spores. Growth Scp160p-GFP temperatures indistinguishable No increase observed encodingGFP-SCP160, theSCP160 −625 +3, 5′-CgagctcGGATCCTTCTTTCTCATTCCTTCATTTAATGTTCG-3′ 5′-CactagtCATTGCAGTTATAATGGAAGGAGGGGG-3′ primers. product subcloned pBlueScript SK+ (Stratagene), pMS403. URA3 pRS314 (31Sikorski R.S. Hieter 1989; 122: 19-27Crossref 5′-GccactccactggGTACTGAGAGTGCACCACGC-3′ 5′-CccagtggagtggGCGGTATTTCACACCGCAGGG-3′ ligated unique BstXI site pMS403 position −425 gene, pMS404. Into SCP160-coding generated YEP13/6 5′-CCatcgatGTCTGAAGAACAAACCGCTATTGAC-3′ 5′-GgtcgacCGATGGAGAATTCAAAATAGATTC-3′ Upstream ATG SCP160, introduced GFP-coding region, pMS356 5′-GactagtAAAGGAGAAGAACTCTTCACTGGAGTTG-3′ 5′-CatcgatCCTTTGTATAGTTCATCCATGCCATGTG-3′, pMS405. linear 4.4-kilobase BamHI-EcoRI digest transformed haploid (32Winston Dollard Ricupero-Hovasse S.L. 53-55Crossref (796) express GFP-tagged Tub1p, pRS303 HIS3 promotor upstream GFP-TUB1 (33Straight Marshall W.F. Sedat J.W. Murray A.W. 277: 574-578Crossref (321) NheI CEN containingSCP160 subcloning aBamHI-PstI pRS315 pMS346. detect immunoblotting procedures, membranes (Protean, Schleicher & Schuell) incubated 2 h room temperature antibodies diluted buffer (PBS, 0.2% Tween 20, 5% dry milk powder) followed peroxidase-conjugated secondary (Sigma) detection ECL (Roche Molecular Biochemicals). Antibodies raised rabbits (New Zealand White) glutathioneS-transferase-tagged expressed, purified fromEscherichia coli antigen. against ribosomal Rps3p Rpl35p rabbits, standard techniques (34Harlow Lane Antibodies: Laboratory Manual. Cold Spring Harbor Laboratory, Harbor, NY1988: 55-138Google peptides VALISKKRKLVADC-CONH2and CPIRKYAIKV-COOH, respectively, conjugated keyhole limpet hemocyanin (Pierce) sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce). Antiserum Kar2p produced (35Rose M.D. Misra L.M. Vogel J.P. 57: 1211-1221Abstract (595) Sec61p have (36Stirling C.J. Rothblatt Hosobuchi Deshaies Schekman 3: 129-142Crossref (280) Zwf1p obtained Sigma. For immunodetection, antibody dilutions follows: (1:2,000 1:10,000), (1:250,000), (1:25,000), (1:10,000), (1:5,000). Yeast grown 30 °C complete (yeast extract/peptone/dextrose) selective medium (Hartwell Complete media (37Adams Gottschling Sterns Methods NY1997Google toA600 = 0.5–0.6. Cycloheximide added concentration 100 μg/ml, cultures another 15 min °C. Cells harvested resuspended 1/100 culture volume ice-cold low-salt (LS) (20 mm Hepes-KOH, pH 7.6, potassium acetate, 5 magnesium 1 EDTA, dithiothreitol, μg/ml cycloheximide, 0.1 phenylmethylsulfonyl fluoride, protease inhibitor mix (according manufacturer; Roche Biochemicals)). After addition two volumes glass beads, lysed vigorous shaking 4 Crude lysates 7–12 mg/ml centrifugation 1,200 × g. fractionated consecutive 6,000, 18,000, 200,000 g 20 each step, pellets rinsed water frozen liquid nitrogen; supernatants subjected next step. Lysates LS buffer, 7 25 units/ml micrococcal S1 nuclease 500 acetate 6,000 ×g min. 150 μl supernatant (corresponding 7.5 A600) loaded 4-ml 10–40% (w/v) sucrose gradient or, if pretreated EDTA. Gradients centrifuged 55,000 rpm SW60 rotor (Beckman) 400-μl fractions ISCO640 fractionator, continuous monitoring absorbance 254 nm. Each fraction precipitated 10% trichloroacetic acid, pellet washed 80% (v/v) acetone, dried, SDS sample buffer. expressing fusion 0.3–0.6 under Olympus BX60 microscope Zeiss UPlanAPO 100× oil immersion objective. Hamamatsu C4742–95 CCD camera Openlab software package (Improvision, Heidelberg, Germany) acquire pictures. Exposure times varied corresponding level 0.5 s (Sec63-GFP) 3 (GFP-Scp160p). analyze cytoskeleton, 0.6 A600 fixed 3.7% formaldehyde h. Spheroplasts prepared transferred onto slides coated 0.02% poly-lysine. 5-min PBS, 0.1% 1% bovine serum albumin, spheroplasts 40 0.66 μm units/ml) AlexaTM488-phalloidin (Molecular Probes) albumin 4′6-diamido-2-phenylindoledihydrochloride (DAPI) PBS mounted glycerol. (28Kruse However, indirect immunofluorescence majority large analyzed respect polysome association. Therefore, whole fractionation clarify ribosomes. entire pool total lysate cycloheximide-treated bead lysis acetate. From lysate, obtained, (P6), ribosomes (P18 P200) ribosome-free (S200). As shown Fig.1, half luminal present P6, other S200, due release result mechanical damage microsomes lysis. Almost marker minor P18. glucose-6-phosphate-dehydrogenase (Zwf1p) only S200. immunodetection small Rpl35p, about P200 fraction. smaller proportion P6 Taken together, indicates membranes, membrane-free (cytosolic) P18 contains amount heavy sedimented membrane-containing (Fig. 1); second, ribosome P200). Ribosome-depleted (S200) almost Scp160p. clearly fractions. Next density gradients. All membrane-depleted comigrated A). Scp160p-containing than monosomes detected. Additional gradients 10 50% accumulation (data shown). our analysis, comigration very labile. processed immediately. cells, reduced, some slower migrating appeared cofractionation salt-sensitive, indicating ionic interaction between top B). Removal Mg2+ ions leads dissociation 80 S partially disassembled unfolded subunits (38Nolan R.D. Arnstein H.R. 1969; 96-101Crossref (15) 39Blobel 1971; 68: 1881-1885Crossref treatment, shifted sedimenting (Fig.2 C, 1–5), peaked fractions2–4, suggesting causes sediments similar subunit. sized particle, formed distinguish contributes directly naked ribosomes, selectively digested mild nuclease. digestion, migrated D, fractions6–9). vast fractions1 2; amounts fractions3–7), Treatment higher concentrations caused rRNA seen 2, C D) corresponds absence agents. membrane-enriched (P6) nonionic detergent Nikkol separated solubilized material unsolubilized recentrifugation Under conditions, (Fig.3 Due during solubilization complete. Solubilized cosedimented high speed min; g; Fig.3 A, lanes 8), bound this, Nikkol-solubilized centrifugation. major peak B, 6) peaks fractions7–10). cofractionated maintained. Some 2–5; however, remained mono- polysome-containing fractions6–10). associates compare treated either control incubation 23 did significant P6. Incubation removal led extraction two-thirds (Fig.4 A); results abolished membrane, again ribosome-mediated membrane. Digestion less efficient. nascent translating puromycin/GTP cause mRNA-dependent, untreated nuclease-treated Half solubilized, 5–10). reduced stay attached (compare and6; Fig. 4, B C). Ribosomes removed. dynamic living fusions created encode bright derivative (40Kahana J.A. Schnapp B.J. 92: 9707-9711Crossref (127) moiety N terminus constructs genome completely making copy cell. approach allowed us study normal levels. An immunoblot probed anti-Scp160p confirmed version (Fig.5 Furthermore, levels tagged forms untagged Fluorescence concentrated around patches close periphery C), reminiscent staining. correlate directly, resident Sec63p. Sec63p-GFP broadly pattern Sec63p-GFP, GFP-Scp160p typical cortical seem excluded nucleus, diffuse cytoplasmic staining visible. partial overlapping integral Sec63p Next, overexpressing plasmid. Quantitation indicated approximately 4-fold overexpression induced redistribution (Fig.6 A), consistent limited number sites. behaves similarly endogenous supernatant, Nikkol, derived empty CENplasmid preferentially 6 panel, 6–10). 2). changed dramatically Scp160p; 2–4 bottom panel). complex, emerged 6–10) behaved Increased 2–4). roughly constant 6, andC, suggest specific, saturable how get ER, disassembled. depolymerized network mi