An Arabidopsis Prolyl 4 Hydroxylase Is Involved in the Low Oxygen Response.

Plant responses to flooding, submergence and waterlogging are important for adaptation to climate change environments. Therefore, the characterization of the molecular mechanisms activated under hypoxic and anoxic conditions might lead to low oxygen resilient crops. Although in mammalian systems prolyl 4 hydroxylases (P4Hs) are involved in the oxygen sensing pathway, their role in plants under low oxygen has not been extensively investigated. In this report, an Arabidopsis AtP4H3 T-DNA knock out mutant line showed higher sensitivity to anoxic treatment possibly due to lower induction of the fermentation pathway genes, ADH and PDC1, and of sucrose synthases, SUS1 and SUS4. This sensitivity to anoxia was accompanied by lower protein levels of AGPs-bound epitopes such as LM14 in the mutant line and induction of extensins-bound epitopes, while the expression levels of the majority of the AGPs genes were stable throughout a low oxygen time course. The lower AGPs content might be related to altered frequency of proline hydroxylation occurrence in the p4h3 line. These results indicate active involvement of proline hydroxylation, a post-translational modification, to low oxygen response in Arabidopsis.

Mitochondrial ATP-independent chaperones.

Mitochondria possess a dedicated-chaperone system in the intermembrane space, the small Tims that are ubiquitous in all eukaryotes from yeast to man. They escort membrane proteins to the outer or the inner membrane for proper insertion. These mitochondrial chaperones do not require external energy to perform their function and have structural similarities to other ATP-independent chaperones. Here, we discuss their structural properties and how these relate to their chaperoning function in the mitochondrial intermembrane space.

Mutation of conserved charged residues in mitochondrial TIM10 subunits precludes TIM10 complex assembly, but does not abolish growth of yeast cells.

The Saccharomyces cerevisiae TIM10 complex (TIM10c) is an ATP-independent chaperone of the mitochondrial intermembrane space, involved in transport of polytopic membrane proteins. The complex is an alpha(3)beta(3) hexamer of Tim9 and Tim10 subunits. We have generated specific mutations in charged residues in the central core domain of each subunit delineated by the characteristic twin CX(3)C motif, and investigated the effect of these mutations on subunit folding, complex assembly and TIM10 function in vitro and in vivo. Any combination of mutations that included a specific glutamate residue, conserved in all known Tim9 and Tim10 sequences, abolished assembly of the TIM10 complex. In vivo complementation analyses using a MET3-TIM10 strain that is selectively inactivated for the expression of wild-type Tim10 showed that (i) an N-terminal deleted version of Tim10 that was previously shown to be defective in substrate binding is lethal under all conditions, but (ii) the charged residues mutant of Tim10 that is defective in assembly with Tim9 can restore growth in glucose, but not in non-fermentable carbon sources. These data suggest that formation of the hexamer is beneficial but not vital for TIM10 function, whilst the N-terminal substrate-binding region of Tim10 is essential in vivo.

A novel intermembrane space-targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding.

Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step-specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys.