Expression systems
For expression in mammalian cells, all β-proteins, S-GFP (NES-NLS-EGFP), and the cytoplasmic, nuclear or mitochondrial targeted versions of Luciferase-EGFP (Gupta 2011) were cloned into pcDNA3.1 Myc-His A vectors (Invitrogen) via KpnI and XbaI. All sequences were followed directly by a stop codon, excluding the expression of any 3’ c-Myc- and His-tags of the vector.
Myc-Htt96Q was cloned into pcDNA3.1 Hygro(+) by Gregor Schaffar (Schaffar 2004).
TDP-F4 was received from the nonprofit repository Addgene (TDP-43-EGFP construct 4; Yang 2010).
ParkinΔC (ParkinW453X) was kindly provided by Dr. Konstanze Winklhofer (Winklhofer 2003).
For bacterial expression (E. coli), the N-terminally c-Myc-tagged β proteins and αS824 were cloned into pTrcHis (IPTG inducible; Invitrogen) or pBad (titratable expression by carbon sources such as arabinose, glucose, glycerol), excluding expression of His-tags included in the vector. For purification, αS824 was cloned into the pProEx HT vector N-terminal of the His tag, which was cleaved off by TEV protease during purification of the protein.
Sequence design
The β proteins and αS824 were fused to an N-terminal c-Myc tag (MCEQKLISEEDLG) directly followed by (Olzscha 2011, West 1999):
β4
MQISMDYQLEIEGNDNKVELQLNDSGGEVKLQIRGPGGRVHFNVHSSGSNLEVNFNNDGGEVQFHMH β17
MQISMDYEIKFHGDGDNFDLNLDDSGGDLQLQIRGPGGRVHVHIHSSSGKVDFHVNNDGGDVEVKMH β23
MQISMDYNIQFHNNGNEIQFEIDDSGGDIEIEIRGPGGRVHIQLNDGHGHIKVDFHNDGGELQIDMH
αS824
MYGKLNDLLEDLQEVLKNLHKNWHGGKDNLHDVDNHLQNVIEDIHDFMQGGGSGGKLQEMMKEFQQVLDELNNHLQGGKHTVHHIEQNIKEIFHHLEELVHR
NES-β4/β17/β23/αS824 were generated by integrating a consensus NES (LELLEDLTLG; la Cour 2004) behind the c-Myc tag of the β proteins and αS824, resulting in (MCEQKLISEEDLELLEDLTLG).
NLS-β4/β17/β23/αS824 were generated by inserting two repeats of the SV40 derived NLS (DPKKKRKV) N-terminal of c-Myc tagged β proteins or αS824, resulting in
(MAADPKKKRKVDPKKKRKVDTYGAGEQKLISEEDLNGASS).
Mito-β4/β17/β23/αS824 I and II were generated by inserting either the mitochondrial leader sequence of human mtHsp60 (MLRLPTVFRQMRPVSRVLAPHLTRAYAKDVKFGCEQKLISEEDLGMQ IS) or of human COX8 (MSVLTPLLLRGLTGSARRLPVPRAKIHSLCEQKLISEEDLG) followed by a c-Myc sequence N-terminal to the β proteins or αS824. Both leader sequences were functional.
To generate EGFP fusion proteins, the N-terminally c-Myc tagged β proteins or αS824 were fused N-terminal to EGFP (amplified from pEGFP-N1), connected via a flexible 16 amino acid long linker sequence (TSGSAASAAGAGEAAA; Chang JMB 2005) and cloned via KpnI and XbaI into pcDNA3.1 Myc-His A (Invitrogen). Any 3’ tags of the vector are not expressed, omitted by a stop codon directly behind the EGFP sequence.
The NLS-β23 (K7T, K15T) and NLS-EGFP (K7T, K15T) nuclear import mutants were generated by site-directed mutagenesis, amplifying the respective NLS plasmids by PCR with an oligonucleotide carrying the nucleobases to be exchanged (Kalderon 1984, Hodel 2001).
S-GFP was generated by inserting the described NLS and NES connected via short linkers with an HA tag (MAADPKKKRKVDPKKKRKVDTYGAGYPYDVPDYALLELLEDLTLSGSDL) N-terminal to EGFP (from pEGFP-N1) via KpnI and XbaI into pcDNA3.1 Myc-His A (Invitrogen), omitting other tags of the vector.
Targeting versions of the β proteins and αS824 and constructs fused to EGFP were created by PCR amplification, fusing oligonucleotides with overlapping sequences and amplifying the fused constructs with short 5’ oligonucleotides for both strands. Integrated KpnI and XbaI restriction sites allowed restriction and ligation into the described vectors. See below for details of the individual steps of cloning.
American firefly luciferase, in form of the wild type protein or including a single or a double point mutation selected for thermal destabilization of luciferase, was fused to EGFP for use as fluorescent proteostasis sensors (Gupta 2011). Here, the luciferase-EGFP proteins were combined with nuclear export signals, nuclear localization sequences, or mitochondrial targeting sequences (as described for the β proteins, excluding the c-Myc tag) to direct the sensors specifically into these compartments.
The targeting signals were fused N-terminally by PCR and ligated into pcDNA3.1 vectors using KpnI and XbaI restriction sites.
Agarose gel electrophoresis
DNA fragments were separated in 0.8-1.5% agarose gels (LE agarose, Biozym) casted in TAE buffer (50x stock per liter: 242 g TRIS base, 57 mL acetic acid, 37 g EDTA), including 0.01% SYBR Safe DNA Gel Stain (Invitrogen). Samples were applied in DNA loading buffer (6x stock solution: 30% glycerol, 0.25% bromophenol blue) with a 1 kb DNA ladder (Promega) and run at 100 V in TAE. For visualizing, the gel was illuminated using blue light (Sage Imager 2.0, Invitrogen).
Polymerase chain reaction (PCR)
For molecular cloning or sequence analysis, DNA fragments were amplified by PCR (Mullis 1986). The components of a standard PCR are described below. In case of inefficiency, concentrations of primers, template, magnesium ions, or the annealing temperature were modified.
The PCR reaction was run including Herculase DNA Polymerase (Agilent Technologies) or Pfu DNA Polymerase (Promega) in T3 Thermocyclers (Biometra). For mutagenesis, the complete plasmids were amplified in 20-25 cycles, syntheses of inserts for cloning or fusion of DNA fragments were performed in up to 30 cycles.
Table 2 | Standard PCR setup.
Reagent Volume
DNA template (50-300 ng/µL) 1 µL
5’-Primer 0.1 µM 5 µL
3’-Primer 0.1 µM 5 µL
dNTPs (2.5 mM each nucleotide) 5 µL
10x Herculase buffer 5 µL
Herculase (5 U/µL) 1 µL
ddH20 added to a final volume of 50 µL
Table 3 | Standard PCR cycling program.
Reaction Step Temperature Time
Initial denaturation 95°C 2 min
Denaturation 95°C 30 sec
Annealing 40-60°C 30-60 sec
Elongation 72°C 1 min/kb
Final Elongation 72°C 10 min
Storage 4°C
∞
PCR products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega).
Site-directed mutagenesis (mutations, insertions, and fusion proteins) Site-directed modifications of genetic sequences were created by PCR.
Site-directed mutations, such as an exchange of single codons in the SV40 nuclear localization signal rendering it functionally ineffective, were introduced by amplifying the complete gene or the entire plasmids with synthetic oligonucleotide primers carrying the desired modifications (exchange of a single or a few nucleotides). Afterwards, the original methylated plasmids were digested by DpnI (NEB) for 60 min at 37 °C, and the remaining solution was added to chemically competent DH5α cells for transformation.
For insertions or deletions of e.g. protein targeting signals or antibody binding sites, similar oligonucleotide primers were applied containing the desired sequences including restriction sites.
Fusion proteins (such as EGFP fusions, introduction of protein targeting signals) were created by amplifying DNA sequences carrying overlapping sequences. Overlapping sequences were therefore introduced running a PCR with primers containing the respective sites at their 5’ or 3’ ends. These PCR products were purified and fused in a second PCR reaction, where the overlapping sequences bind to each other so that DNA polymerase can prolong residual complementary sequences.
Additional short primers at the 5‘ ends of the complete fusion sequence allowed amplifying the entire fused sequences selectively in the same reaction. The fusion sequences were then purified, restricted and ligated into desired vectors.
Molecular cloning (restriction, ligation)
The restriction of plasmids and linear DNA inserts was performed applying site-specific restriction enzymes (NEB or Fermentas). Restricted plasmid backbones were dephosphorylated to prevent self-religation (Calf Intestinal Alkaline Phosphatase, NEB). Concentrations, buffers, and incubation times were chosen according to the manufacturers’ instructions or modified, if necessary. Results were controlled by agarose gel electrophoresis. DNA fragments of interest were selected and purified for further processing (Wizard SV Gel and PCR Clean-Up System, Promega).
For ligation, 0.5 µL of T4 DNA ligase (Promega) was added to a total volume of 30 µL reaction mix, containing 50-200 ng restricted vector, 3-7 times (molar ratio) restricted inserts, and ligation buffer (Promega). Restrictions were carried out overnight at 16 °C under agitation. Up to 5 µL of the ligated sample were added to chemically competent DH5α cells for transformation.
Preparation and transformation of chemically competent E. coli cells
An appropriate E. coli strain (DH5α for plasmid preparations or BL21 (DE3) for protein purification) was plated out on LB agar. From this, a single colony was grown in a small pre-culture to inoculate 1 L LB medium growing to an OD600 of < 0.5. Cells were centrifuged at 6,000 x g for 10 min at 4 °C and resuspended in ice-cold sterile Ca2+/glycerol solution (60 mM CaCl2, 10 mM Pipes, 15% glycerol, pH 7.0). After a second centrifugation and resuspension, cells were kept on ice for 30 min. A third centrifugation followed, cells were resuspended in 10 mL ice-cold Ca2+/glycerol buffer and dispensed in e.g. 200 µL aliquots. Cells were immediately frozen in liquid nitrogen and stored at -80 °C (adapted from Davis 1994).
For transformation, 50 µL chemically competent cells per sample were thawed on ice, and 10-500 ng plasmid DNA or up to 5 µL of a ligation reaction mix were added. After incubation on ice or at 4 °C for 20 min, cells were heat shocked at 42 °C for 45 sec and cooled on ice for further 5 min. 1 mL of LB medium was added, and the cells were incubated at 37 °C shaking for 1 h. Cells were sedimented and 400 µL of fresh LB medium was added. The cells were plated on LB agar plates containing antibiotics for selection (100 µg/mL ampicillin or 50 µg/mL kanamycin) and incubated overnight at 37 °C.
Plasmid preparation
Plasmids were prepared from selective LB cultures starting from a single colony of DH5α cells transformed recently.
Plasmids for bacterial transformation or cloning applications were prepared using the Wizard Plus SV Minipreps kit (Promega). Endotoxin free plasmids for human cell culture were prepared utilizing the EndoFree Plasmid Maxi Kit (Qiagen) according to the supplier’s instruction.
Concentration and quality of purified DNA was determined by UV spectroscopy (260/280 nm), and sequences were confirmed by DNA sequencing (core facility, MPI of Biochemistry).