L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis

Abstract

L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.

Keywords: Ascorbate biosynthesis; Complex I; Complex I assembly; GLDH; Plant mitochondria; Respiratory chain.

Fig. 1

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

Fig. 2

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

Fig. 3

Localisation of GLDH by two-dimensional BN-SDS-PAGE. Mitochondrial complexes of Col-0 and the complex I mutant ndufs4 were resolved by BN-SDS-PAGE. Duplicate gels were run and either stained with Coomassie (top panels) or transferred onto a membrane for western blot analysis with anti-GLDH (middle panels) or anti-CA (bottom panels) antibodies. The positions of complexes I, III and V of the OXPHOS system are indicated above the top panel. The positions of complex I and its assembly intermediates are indicated above of the central panels. Complexes not detected in the western blots are indicated in grey to facilitate comparison between the two samples

Fig. 4

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

Fig. 5

Model showing the different steps of complex I assembly. GLDH is indicated in black and complex I assembly intermediates are shown in grey. The name of each assembly intermediate is indicated above each complex (except for the 650 kDa intermediate). The localisation of the carbonic anhydrases in the complex is indicated by CA. In Col-0, GLDH is incorporated during the transition between the 200 and 400 kDa intermediates, and released when complex I* is converted into complex I. In the ndufs4 mutant, the matrix arm is not assembled, leading to the full assembly of the membrane arm (intermediate of 650 kDa) after the release of GLDH