Anti-GLDH | Galactono-1,4 lactone dehydrogenase

Submitochondrial localization of GLDH. Purified mitochondria (tot) were fractionated and the following submitochondrial compartments were isolated: matrix (ma), inner membrane (IM), intermembrane space (IMS) and outer membrane (OM). Integral proteins of IM (IMi) and peripheral proteins of IM (IMp) were separated by performing an alkali treatment of the IM (see “Material and methods”). In addition, mitochondria were swollen to obtain mitoplasts (swollen mitochondria lacking the outer membrane). Mitoplasts (mt?) were treated with proteinase K to digest the protein domains of the IM facing the IMS (mt+). The different fractions were separated by SDS-PAGE and western blots using anti-GLDH antibodies and antibodies against marker proteins for each compartment were performed

Localisation of GLDH by BN-PAGE. Mitochondrial complexes of Col-0 and the complex I mutant ndufs4 were resolved by Blue-Native PAGE. Duplicate gels were run and either stained with Coomassie (left panel) or transferred onto a membrane for western blot analysis with anti-GLDH (middle panel) or anti-CA (right panel) antibodies. The position of selected respiratory complexes is indicated on the left. The size (in kDa) of complex I and the different assembly intermediate of complex I is indicated on the right

Characterisation of complex I assembly in the gldh mutant. Total membranes were purified from Col-0 and the ascorbate-deficient mutants gldh and vtc2-1. a BN-PAGE analysis of membrane complexes. The left panel shows the Coomassie staining of the gel. The right panel shows the western blot analysis using anti-CA antibodies. The sizes of the marker bands of the molecular weight marker used for calibration of the gel are indicated in kDa on the left. The positions of complex I (I), complex I* (I*), photosystem 1 (PS1), photosystem 2 (PS2), the LHC trimers (LHC3) and LHC monomers (LHC) are indicated between both panels. b BN-SDS-PAGE analysis of membrane complexes. Top panel Coomassie staining of a representative gel (obtained for Col-0). The positions of photosystem 1 (PS1), photosystem 2 (PS2), the LHC trimers (LHC3) and LHC monomers (LHC) are indicated on the top. The bottom three panels show western blot analyses using anti-CA antibodies. The plant line analysed is indicated on the right. The positions of complex I (I) and the assembly intermediates detected in the gldh mutant are indicated in the bottom of the panel