using protein scaffold vs carrier protein

Cryosections were picked up using a drop of solution containing 1% methyl cellulose and 1.15 M sucrose and thawed in PBS. Infiltrated samples were immersed in liquid nitrogen and cryosectioned using a Leica EM FCS ultramicrotome (Leica) equipped with a cryochamber (Ultracut Leica) at −100☌. Slices of gelatin-embedded pellet were immersed in 2.3 M sucrose and left for 6 days on a rotating wheel at 4☌. The pellet was resuspended in 10% gelatin dissolved in water at 37☌, centrifuged, and solidified on ice. After centrifugation at 1,300 × g for 10 min, cells were washed three times in 0.1 M phosphate buffer containing 0.01 M glycine.

Digitonin fractionation and Western blot analysis.Ĭells were fixed either with 4% formaldehyde or 4% formaldehyde with 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 1 h at room temperature. Finally, the samples were mounted with Antifade reagent with 4′,6′-diamidino-2-phenylindole (DAPI Invitrogen) and after incubation in the dark for 30 min were visualized using an Axioplan2 imaging fluorescence microscope (Zeiss).

Slides were washed with PBS prior to the application of Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) at a 1:1,000 dilution for 1 h at room temperature. Next, primary anti-v5 or anti-HA 3 antibodies (Invitrogen) were added at a 1:100 dilution in PBS-Tween (0.05%) with 5% milk (Laktino, low fat PML, Czech Republic) and incubated at 4☌ overnight. The slides were washed with PBS, incubated in ice-cold methanol for an additional 20 min, and briefly washed with PBS. The suspension (250 μl) was applied to a microscopic slide and incubated for 10 min at room temperature. After 30 min incubation at 27☌ or 37☌, the cells were centrifuged for 5 min at 1,300 × g at room temperature, and the pellet was washed and resuspended in 1 ml phosphate-buffered saline (PBS) with 4% formaldehyde and 1.25 mM NaOH. Complex V (ATP synthase) is present, but its F1 part rotates in the reverse direction compared to the PS, cleaving ATP and pumping H + out of the mitochondrion in order to create the membrane potential required for protein import ( 2).Īpproximately 1 × 10 7 PS or BS cells were collected, centrifuged, resuspended in fresh medium, and stained with a 200 nM or 20 nM concentration, respectively, of the mitochondrion-selective dye Mitotracker red (Invitrogen). Moreover, complex I is expressed, yet its enzymatic activity is not significant, and its real function remains elusive ( 12 – 15). While cytochrome c-dependent respiratory complexes III and IV are altogether missing from the BS, complex II probably remains active. Instead of the canonical respiratory chain, glycerol-3-phosphate dehydrogenase supplies electrons to trypanosome alternative oxidase as a terminal electron acceptor ( 11). In contrast to a typical mitochondrion, the BS organelle does not serve as the main energy source. Ultimately, the import of hundreds of nucleus-encoded mitochondrial proteins involved in protein import and processing, kDNA replication and maintenance, kRNA transcription and processing, and translation are indispensable in the BS ( 3, 4, 9, 10). In addition, since no tRNAs are encoded in the mitochondrial DNA (kinetoplast DNA ), their import from the cytosol and modifications are mandatory for mitochondrial translation ( 4, 8). Since the A6 transcript undergoes extensive RNA editing, the entire RNA processing machinery is active and essential in the BS as well ( 6, 7).

Hence, translation is indispensable ( 4, 5). Out of all 14 mitochondrial gene products, only subunit A6 of ATPase is translated and utilized in the BS as an indispensable part of respiratory complex V, which is present and active, even though it does not fulfill its standard function ( 2, 3).

Using protein scaffold vs carrier protein