Ni-NTA purification of GCDH His₆-fusion protein.

<p>(<b>A</b>) The expression of recombinant GCDH-His₆ in <i>E. coli</i> was induced by the addition of IPTG (lane 1: before induction, lane 2: after induction). After 4 h <i>E. coli</i> cells were lysed by sonication (lane 3) and centrifuged (lane 4: pellet with insoluble proteins; lane 5: supernatant with soluble proteins). The supernatant with GCDH-His₆ was incubated with Ni-NTA agarose and loaded on a column. Unbounded proteins (lane 6) were removed and the column was washed with increasing imidazole concentrations (lane 7: 10 mM imidazole; lane 8: 50 mM imidazole). Finally the GCDH-His₆ protein was eluted in four steps with increasing imidazole concentrations (lane 9–11: 150 mM imidazole; lane 12: 250 mM imidazole). Samples were separated by SDS-PAGE (10% acrylamide) and proteins were visualized by Coomassie Blue staining. The positions of molecular mass marker proteins (in kDa) are indicated. (<b>B</b>) Validation of the purified GCDH-His₆-fusion protein. Different amounts of purified GCDH-His₆ protein were separated by SDS-PAGE (10% acrylamide) and analyzed by anti-GCDH western blotting. Representative pictures of n = 10 independent preparations are shown.</p

Interaction of GCDH with ETFB <i>in vivo</i>.

<p>(<b>A</b>) Schematic representation of GCDH and ETFB fusion proteins with YFP1 (dark blue) and YFP2 (red). The 10-amino acid linker (GGGGS)₂ is indicated in green. The calculated molecular masses of the respective fusions proteins are shown in brackets. (<b>B</b>) The expression of the various fusion proteins used in this study were analyzed by western blotting. The 43 kDa band (*) reactive with the anti-ETFB antibody is unspecific. (<b>C</b>) Fluorescence microscopy of fixed BHK cells co-expressing GCDH and ETFB fusion proteins showed a strong YFP fluorescence. Nuclei were visualized using DAPI (blue). Scale bars = 40 µM. Representative images of n = 3 independent transfection experiments are shown.</p

GCDH affinity chromatography of mitochondrial matrix extracts.

<p>Purified GCDH was covalently coupled to Affi-Gel 10 matrix and incubated with isolated mitochondrial matrix proteins. Aliquots of the loaded extract (input, I: 10% of total protein), the last wash fraction (W, 25%) and the high salt elution fraction (E, 100%) were separated by SDS-PAGE (10% acrylamide) and tested by anti-DLST (<b>A</b>) and anti-ETFB (<b>B</b>) western blotting. Non-coupled Affi-gel 10 beads were used as a control for unspecific binding. The positions of the molecular mass marker proteins (in kDa) are given. A representative blot of n = 3 independent preparations is shown.</p

Mitochondrial proteins binding to GCDH.

<p>GCDH-His₆ was immobilized on beads and incubated with isolated mitochondrial matrix proteins from pig liver. The identity of specifically co-purifying proteins was determined by LC-MS/MS.</p><p>M: mitochondrial matrix; IM: inner mitochondrial membrane.</p

YFP fragment complementation assay demonstrates the interaction of GCDH with DLST <i>in vivo</i>.

<p>(<b>A</b>) Schematic composition of C-terminal YFP1 (dark blue) and YFP2 (red) fusion proteins of DLST, GCDH and MCFD2 used in this study. The 10-amino acid linker (GGGGS)₂ is indicated in green. The calculated molecular masses of the fusion proteins are shown in brackets. The ERGIC marker protein MCFD2-YFP2 was used as negative control. (<b>B</b>) Expression analysis in BHK cells of all fusion proteins visualized by western blotting, using anti-DLST, anti-GCDH and anti-GFP antibodies. (<b>C</b>) Fluorescence microscopy of the indicated single or co-expressed fusion proteins. Strong YFP fluorescence was observed in cells co-expressing either GCDH-YFP1 with DLST-YFP2 or GCDH-YFP2 with DLST-YFP1. Nuclei were visualized using DAPI (blue). Scale bars = 40 µM. Representative images of n = 3 independent transfection experiments are shown.</p

Co-Precipitation of DLST with GCDH.

<p>Extracts from HeLa cells overexpressing the DLST-His₆ alone (DLST) or together with GCDH-Myc (DLST+GCDH) were incubated with Ni-NTA agarose for 4 h. Aliquots of the cell extract (input, I: 10% of total), the unbound protein supernatant after precipitation of Ni-NTA beads (S, 10%), the last wash (W, 25%) and the eluted fraction (E, 100%) representing bound proteins, were analyzed by successively exposing the blot to anti-GCDH and, after stripping, to anti-DLST antibodies. Extracts of HeLa cells overexpressing DLST-His₆ and LC3-GFP (DLST+LC3) were used as negative control and analyzed by anti-LC3 western blotting. The expression of DLST was analyzed by anti-DLST western blotting. The position of the 40 kDa molecular mass marker protein is indicated. The figure shows a representative blot of n = 3 independent experiments.</p

PrP^C-GPIThy-1 is glycosylated and transported to the plasma membrane.

<p>(A) Schematic presentation of GPI-anchored PrP^CWT and the PrP^C fusion protein with the GPI-anchor of Thy-1 (PrP^C-GPIThy-1). The substitution of the GPI-anchor signal sequence (ss) of the PrP for the one of Thy-1 is indicated. (B) Western blots of PrP^CWT and PrP^C-GPIThy-1 stably expressed in MDCK cells. A clone with a similar expression level as PrP^CWT was chosen. The glycotype of di-, mono-, and non-glycosylated PrP^C-GPIThy-1 is unchanged. (C) Assessment of non-permeabilized membrane localization of PrP^CWT and PrP^C-GPIThy-1 by confocal microscopy shows plasma membrane localization of both proteins (scale bar is 10 µm). (D) Sucrose density gradient centrifugation of 1% Triton-X100 extraction at 4°C of PrP^CWT and PrP^C-GPIThy-1 cells reveal localization of both in flotillin enriched DRMs. Fractions were taken from the top (fraction 1) to the bottom (fraction 12).</p

Thy-1-GPI anchor redirects PrP^C to the apical site.

<p>(A) Cells stably expressing PrP^CWT and PrP^C-GPIThy-1 were grown in Transwells for 4 to 5 days, processed for immunocytochemistry, and analyzed with confocal microscopy. YZ sections (left) and view on the membrane (right) at the level of tight junctions stained for ZO-1 (red) confirm both polarization and confluency of cells and show increased apical signal for PrP^C-GPIThy-1 (green). (B) After staining with PrP 3F4 antibody under non-permeabilizing conditions, serial Z-stacks from the bottom to the top were taken. YZ sections show transversal cut through cells at the level of the dashed line in mid. PrP^C-GPIThy-1 was found at the apical membrane when compared to PrP^CWT. Scale bars are 10 µm. (C) Cells grown in Transwells labeled with EZ-Link Sulfo-NHS-SS-Biotin either apically (a) or basolaterally (b) were processed for Western blotting for PrP^C and E-Cadherin (as control of cell polarization) in parallel. The graph (three independent experiments) shows mean percentages ± SEM of apical (a) or basolateral (b) amount of protein when compared to the total amount which is set at 100%.</p

Schematic drawing of constructs used in this study.

<p>Shown are the maps of PrP^CWT, PrP^CG1, PrP^CG2, and PrP^CG3 with N-terminal signal sequence (ss) and C-terminal GPI-anchor signal (ss GPI-anchor) (dark boxes) and the mutations introduced to delete N-gylcosylation sites.</p

Lentiviral vectors and immunoblot analyses of culture supernatants from clonal CNTF-NS and control-NS cell lines.

<p>A lentiviral vector encoding a secretable variant of mouse ciliary neurotrophic factor (CNTF), an internal ribosome entry site (IRES) sequence of the encephalomyocarditis virus and a Venus reporter and a zeocin (ZEO) resistance gene separated by a P2A sequence of porcine teschovirus-1 (2A) under regulatory control of the cytomegalovirus enhancer/chicken ß-actin (CAG) promoter (a) was used to generate CNTF-secreting NS cells. NS cells for control experiments were transduced with a vector containing the CAG promoter, an IRES sequence and a tdTomato (tdTom) reporter gene fused to a blasticidin (BSD) resistance gene (b). Immunoblot analysis (c) of culture supernatants from the newly established CNTF-NS cell clone (clone 2) revealed elevated secretion levels of CNTF when compared to the original clonal CNTF-NS cell line (clone 1). Supernatants from control-NS cell clones (control) lacked detectable levels of the cytokine (c). Recombinant mouse CNTF (rmCNTF) was loaded as a reference. Ψ, packaging signal; cPPT, central polypurine tract; LoxP, recognition site of Cre recombinase; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element.</p