6.1.1 Chemicals
All chemicals used in this study were analytical grade and purchased from Sigma-Aldrich, Merck or Carl Roth if not indicated otherwise. Enzymes and endonucleases were purchased from New England Biolabs.
6.1.2 Growth media and general buffer
Yeast media: Yeast cells were grown in YPD media (1 % (w/v) bacto yeast extract, 2 % (w/v) bacto peptone, 2 % (w/v) dextrose) or in defined synthetic complete media (SC) (0.67 % (w/v) bacto yeast nitrogen w/o amino acids, 2 % (w/v) dextrose, 0.2 % (w/v) amino acid dropout mix) lacking distinct amino acids. For plates, media were supplemented with 2 % (w/v) agar. Plates were supplemented as indicated with 0.8 M NaCl, 0.75 $g/ml L-Canavanine or 25 $g/ml Hygromycin B.
Amino acid dropout mix: Different kinds of dropout mixes were generated including all amino acids w/o one or more specific amino acid for selection of transformed yeast cells (e.g. Leu, Ura). 10 g of leucine were mixed with 2 g of all remaining amino acids and supplemented with 0.5 g adenine, 2 g uracil, 2 g myo-inositol and 0.2 g p-aminobenzoic acid.
Bacterial media: Bacterial cells were grown in LB media (1 % (w/v) bacto tryptone, 0.5 % (w/v) bacto yeast extract, 0.5 % (w/v) NaCl) which were supplemented with 2 % (w/v) agar for plates.
General buffer:
- 6x DNA loading dye: 30 % (v/v) glycerol, 60 mM EDTA, 1.2 mg/ml bromophenolblue, 1.2 mg/ml xylene cyanol - 5x Laemmli buffer (sample buffer): 250 mM Tris HCl pH 6.8, 10 mM EDTA, 5 % (v/v) SDS, 5 % (v/v) beta-mercaptoethanol, 50 % (v/v) glycerol, 7 mg/ml bromophenolblue
- 4x Stacking gel buffer (SDS-PAGE): 0.5 M Tris HCl pH 6.8, 0.4 % (v/v) SDS - 4x Running gel buffer (SDS-PAGE): 1.5 M Tris HCl pH 8.8, 0.4 % (v/v) SDS - 3.5x Gel buffer (Bis-Tris-PAGE): 1.25 M Bis-Tris HCl pH 6.5
- Tricine gel buffer: 3 M Tris HCl pH 8.45, 0.3 % (v/v) SDS
- 10x SDS running buffer: 25 mM Tris, 20 mM glycine, 1 % (w/v) SDS
- 5x Bis-Tris running buffer (MOPS): 250 mM MOPS, 250 mM Tris-Base, 5 mM EDTA, 0.5 % (v/v) SDS - Cathode running buffer (Tricine gel): 0.1 M Tris, 0.1 M Tricine, 0.1 % (v/v) SDS
- Anode running buffer (Tricine gel): 0.2 M Tris HCl pH 8.9
- 10x Western blot transfer buffer: 25 mM Tris, 192 mM glycine, 0.02 % (w/v) SDS, 20 % (v/v) methanol - 10x TBS buffer: 75 mM Tris HCl pH 8.0, 150 mM NaCl
- 10x TBS-T buffer: 75 mM Tris HCl pH 8.0, 150 mM NaCl, 1 % (v/v) Tween-20 - Ponceau S solution: 0.2 % (w/v) Ponceau S, 5 % (v/v) acetic acid
- ECL solution: (a) 0.1 M Tris HCl pH 8.6, 0.25 mg/ml Luminol; (b) 1.1 mg/ml p-cumaric acid in DMSO; (c) 30 % (v/v) H2O2; for usage mix a:b:c = 1000:100:1
- Phusion polymerase buffer: 10 mM Tris HCl pH 8.8, 50 mM KCl, 2 mM MgCl2, 0.1 % (v/v) Triton X-100 - 50x TAE buffer: 2 M Tris acetate, 50 mM EDTA pH 8.0
- Coomassie blue staining solution: 0.6 % (w/v) Coomassie R250, 50 % (v/v) ethanol, 10 % (v/v) acetic acid - Destaining solution: 50 % (v/v) ethanol, 10 % (v/v) acetic acid
- TFB1 buffer: 30 mM KAc, 50 mM MgCl2, 100 mM KCl, 10 mM CaCl2, 15 % (v/v) glycerol - TFB2 buffer: 10 mM MOPS pH 7.0, 75 mM CaCl2, 10 mM KCl, 15 % (v/v) Glycerol
Materials & Methods
" ,("
6.1.3 Antibiotics, inductors and inhibitors
Antibiotics:
- Ampicillin (Amp): 100 mg/ml stock in ddH2O; working solution: 100 $g/ml (AppliChem) - Kanamycine (Kan): 50 mg/ml stock in ddH2O; working solution: 50 $g/ml (Carl Roth) - Chloramphenicol (Cm): 25 mg/ml stock in 70 % ethanol; working solution: 25 $g/ml (Serva)
Inductors:
- IPTG (Isopropyl-D-thiogalactopyranoside): 1 M stock in ddH2O; 1 mM working concentration (Carl Roth)
Inhibitors:
- PMSF (Phenylmethylsulfonylfluoride): 100 mM stock in isopropanol; 1 mM working concentration (Carl Roth) - TmComplete inhibitor cocktail (Roche)
- Aprotinin, Leupeptin, Pepstatin A (Genaxxon BioScience)
6.1.4 Enzymes and antibodies
Enzymes:
- Shrimp alkaline phosphatase (SAP): 1 U/ml (Fermentas) - T4 DNA ligase: 1 U/ml (Fermentas)
- T7 RNA polymerase: 20 U/ml (Fermentas)
- Phusion DNA polymerase: 2.2 mg/ml (lab purification) - Kapa HiFi polymerase: 1 U/$l (Qiagen)
- TEV protease: 2.3 mg/ml (lab purification) - His-Ulp1: 1.95 mg/ml (lab purification) - Apyrase: 50.000 mU/ml (New England Biolabs)
- Restriction enzymes & CutSmart Buffer (New England Biolabs) - Zymolyase-20T: 20.000 U/g (MP Biomedicals)
- Puromycin: 50 mg/ml (Sigma) - Trypsin / Chymotrypsin (Sigma)
Antibodies:
Primary and secondary antibodies were used 1:10.000 diluted in TBS-T if not indicated otherwise. Polyclonal antibodies against Ssb1, NAC, Zuo1, Ssz1, Rpl4A, Rpl17A and Rps3 are already described elsewhere (KOPLIN et al., 2010). The antibodies Pgk1 (Novex Life Technologies), CBD (Millipore; diluted 1:1000), Nop2 (Roche; diluted 1:1000), GFP (Roche), FLAG (Sigma) as well as the HRP-coupled secondary antibodies (rabbit, mouse; Dianova) are commercially available. The same applies to HRP-Streptactin (Thermo Scientific) used for biotin detection. Antibodies used for C. elegans studies: NAC was already described (KIRSTEIN-MILES et al., 2013), Rpl10 (Biomol), Sec61-alpha (Santa Cruz).
6.1.5 Dyes, kits and markers
Dyes:
- DAPI (Thermo scientific) - Midory Green (Nippon Genetics) - Coomassie brilliant blue R250 (Carl Roth)
Kits:
- Plasmid preparation: DNA spin (Intron) - Gel extraction: MEGAquick (Intron)
"
,)"
- Yeast genomic DNA preparation: YeaStar (Epigenetics) - Northern blotting: Dig Northern starter kit (NKS, Roche) - RNA extraction: RNeasy mini kit (Qiagen)
- Peptide biotinylation: NZ-link NHS-biotin (Thermo Scientific)
Markers:
- 1 kb DNA ladder (Fermentas)
- 100 bp DNA ladder (Peqlab Biotechnology GmbH) - Prestained protein ladder (Fermentas)
- RiboRuler high/low range RNA ladder (Fermentas)
6.1.6 Primer
Materials & Methods
"
Plasmid name Inserted gene Promoter Marker
(S.c./E.c.) Backbone Reference
Ssb1_KRR-AAA Ssb1_KRR428-AAA Ssb1 URA3 / Amp pRS316 this study
Ssb1_KRR-KSE Ssb1_KRR428-KSE Ssb1 URA3 / Amp pRS316 this study
Ssb1_KRKR-AAAA Ssb1_KR603AA-K608A-R613A Ssb1 URA3 / Amp pRS316 this study Ssb1_MQ409TA-F444I Ssb1_MQ409TA-F444I Ssb1 URA3 / Amp pRS316 this study
yEGFP yEGFP Ssb1 URA3 / Amp pRS315 lab collection
yEGFP-Ssb1 yEGFP-Ssb1 Ssb1 URA3 / Amp pSR316 this study
yEGFP-Ssb1#NES yEGFP-Ssb1!574-87 Ssb1 URA3 / Amp pRS316 lab collection
yEGFP-Ssb1#601-13 yEGFP-Ssb1!601-13 Ssb1 URA3 / Amp pSR316 this study
yEGFP-Ssb1#1-600 yEGFP-Ssb1!1-600 Ssb1 URA3 / Amp pSR316 this study
yEGFP-Ssb1#1-509 yEGFP-Ssb1!1-509 Ssb1 URA3 / Amp pSR316 this study
NLS-yEGFP-Ssb1#NES NLS-yEGFP-Ssb1!574-87 Ssb1 URA3 / Amp pRS316 this study NLS-yEGFP-Ssb1#NES
(low)
NLS-yEGFP-Ssb1!574-87 Met17 URA3 / Amp pRS316 this study
yEGFP-Ssb1(low) yEGFP-Ssb1 Met17 URA3 / Amp pRS316 this study
Ssb1 (low) Ssb1 Met17 URA3 / Amp pRS316 this study
yEGFP (low) yEGFP Met17 URA3 / Amp pRS316 this study
NLS-yEGFP (low) NLS-yEGFP Met17 URA3 / Amp pRS316 this study
FLAG-Ssb1#1-509 FLAG-Ssb1!1-509 Ssb1 URA3 / Amp pSR316 this study
Ssb1#1-509-FLAG Ssb1!1-509-FLAG Ssb1 URA3 / Amp pRS316 this study
Rpl25-GFP Rpl25-GFP Rpl25 URA3 / Amp pRS426 KOPLIN et al.,
2010
Nup120-mCherry Nup120-mCherry Nup120 LEU2 / Amp pRS315 lab collection
pSUMO-Ssb1 His6-Smt3-Ssb1 T7 - / Kan pET24 lab collection
pSUMO-Ssb1#601-13 His6-Smt3-Ssb1!601-13 T7 - / Kan pET24 this study
pSUMO-Ssb1#606-13 His6-Smt3-Ssb1!606-13 T7 - / Kan pET24 this study
pSUMO-Ssb1#610-13 His6-Smt3-Ssb1!610-13 T7 - / Kan pET24 this study
pSUMO-Ssb1#NES His6-Smt3-Ssb1!574-87 T7 - / Kan pET24 this study
pSUMO-Ssb1_KRR-AAA
His6-Smt3-Ssb1_KRR428AAA T7 - / Kan pET24 this study pSUMO-Ssb1_KRR-KSE His6-Smt3-Ssb1_KRR428KSE T7 - / Kan pET24 this study
pSUMO-NAC (C.e.) His6-Smt3-icd-1_icd-2 T7 - / Amp pET24 lab collection
pSUMO-NAC-RRK-AAA (C.e.) His6-Smt3-icd-1-RRK29AAA_
icd-2 T7 - / Amp pET24 lab collection
Materials & Methods
" ,,"
6.1.8 E. coli strains
Strain name Genotype Reference
DH5 alpha Z1 laciq, PN25-tetR, Spr, deoR, supE44, !(lacZYA-argFV169), Phi80 lacZ !M15 Invitrogen BL21 (DE3) F-, ompT, hsdSB, (rB-mB-), gal, dcm, rne131, (DE3) Invitrogen XL1-blue ecA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, [F " proAB lacIq Z!M15
Tn10 (Tetr)]) Invitrogen
6.1.9 S. cerevisiae strains
Strain name Genotype Reference
BY4741 MATa, his3!1, leu2!0, met15!0, ura3!0 Euroscarf
DS10 MATa, lys1 lys2 leu2-3,112 his3-11,15 ura3-52 trp1! GAL2+ Euroscarf
nac! BY4741; egd1::hisMX6; btt1::phleo; egd2::leu2 KOPLIN et al., 2010
ssb1,2! BY4741; ssb1::kanMX4; ssb2::nat1 KOPLIN et al., 2010
nac!ssb1,2! BY4741; egd1::hisMX6; btt1::phleo; egd2::leu2; ssb1::kanMX4;
ssb2::nat1 KOPLIN et al., 2010
nac!zuo1! BY4741; egd1::hisMX6; btt1::phleo; egd2::leu2; zuo1::kanMX4 KOPLIN et al., 2010
zuo1! BY4741; zuo1::kanMX4 lab collection
ssz1! BY4741; ssz1::nat1 lab collection
ssz1!zuo1! BY4741; ssz1::nat1; zuo1::kanMX4 lab collection
ssb1,2!ssz1!zuo1! DS10; ssb1::his3; ssb2::leu2; ssz1::nat1; zuo1::kanMX4 lab collection
tif4631! BY4741; tif4631::leu2 this study
ssb1,2!tif4631! BY4741; ssb1::kanMX4 ssb2::nat1; tif4631::leu2 this study
puf6! BY4741; puf6::kanMX4 Euroscarf
sac3! BY4741; sac3::kanMX4 Euroscarf
snu66! BY4741; snu66::kanMX4 Euroscarf
Ssb1-TAP BY4741; Ssb1_TAP_his3MX6 Thermo scientific
Ssb1-TAP, ssz1!zuo1! BY4741; Ssb1_TAP_his3MX6; ssz1::nat1; zuo1::kanMX4 this study
Pwp2-TAP BY4741; Pwp2_TAP_ his3MX6 Thermo scientific
Rix1-TAP BY4741; Rix1_TAP_ his3MX6 Thermo scientific
Ipi1-TAP BY4741; Ipi1_TAP_ his3MX6 Thermo scientific
Nop7-TAP BY4741; Nop7_TAP_ his3MX6 Thermo scientific
Nog2-TAP BY4741; Nog2_TAP_his3MX6 Thermo scientific
APPY-C SALLQSRLLLSAPRRAAATARYC PFUND et al., 2001
P2-beta-C GSLEEIIAEGQKC this study
Rpl14-C GKLAAIVEIIDQKKC this study
r-peptides diverse sequences, resulting from random r-protein digestion this study
NRLLLTG-C SAGNRLLLTGGC STEVENS et al., 2003
sigma32-Q132-Q144-C QRKLFFNLRKTKQC MAYER et al., 2000