L-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana

Gustavo E. Gergoff Grozeff,Hans-Peter Braun,Peter Schertl,Stephanie Sunderhaus,Carlos G. Bartoli,Jennifer Klodmann

doi:10.1074/jbc.m111.305144

Abstract

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

Highlights

L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the final step of the L-ascorbate biosynthesis pathway and at the same time is essential for complex I accumulation
This signal represents the monomeric form of GLDH, which was reported previously [36]; see the two-dimensional blue native (BN)/SDS-PAGE GelMap of Arabidopsis mitochondria
Using native gel electrophoresis procedures, which are combined with in gel activity stains and immunoblotting, novel insights into GLDH localization in plant mitochondria were achieved: (i) GLDH forms part of three mitochondrial protein complexes of 850, 470, and 420 kDa; (ii) all three complexes exhibit GLDH activity upon analysis by a newly developed in gel GLDH activity assay; (iii) the 850-kDa complex represents a known smaller version of complex I [13, 22, 26], here termed complex I*, and the 470- and 420-kDa complexes representing subcomplexes of complex I; (iv) 850-kDa complex I* has a complete peripheral arm; and (v) The GLDH containing 470 kDa complex represents the membrane arm of complex I*, which represents a large fragment of the membrane arm of the holoenzyme

Summary

Background

L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the final step of the L-ascorbate biosynthesis pathway and at the same time is essential for complex I accumulation. Complex I has a molecular mass of 1000 kDa and in plants includes at least 48 different subunits, several of which represent proteins specific for this enzyme complex in plants [23, 24] Some of these extra subunits integrate side activities into this respiratory complex, e.g. carboanhydrase (CA) subunits, which were proposed to. GLDH was not found to be attached to the 1000-kDa holoenzyme, but only to a slightly smaller version of complex I, which is of comparatively low abundance [13, 22, 26] This complex has a molecular mass of about 850 kDa and obviously lacks some of the subunits present in the main form of complex I. We propose that GLDH has a more extended function in complex I assembly by binding to several of its assembly intermediates

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION