Biology subjects

Homagk, M.

Publications and source records attributed to Homagk, M..

3 recordsLinked to original sources

bioRxiv2023

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC1 and GRXC2 being better reductants than GRXC3 and GRXC4 with bis(2-hydroxyethyl) disulfide and oxidized roGFP2 as substrates. Vice versa, GRXC3 and GRXC4 are better oxidants of reduced roGFP2 in the reverse reaction. Analysis of electrostatic surface potentials mirrors the phylogenetic classification of class I GRXs but cannot fully account for the observed kinetic differences in their interaction with roGPF2. Despite localization of two class I GRXs each in the cytosol and the endomembrane system, the respective double null mutants are viable without obvious phenotypes. Summary statementWe identify Arabidopsis glutaredoxins GRXC3 and GRXC4 as type II membrane proteins in the secretory pathway and GRXC1 as attached to membranes through N-terminal myristoylation. Cytosolic GRXC1 and GRXC2 and luminal GRXC3 and GRXC4 display distinct biochemical properties in their redox activities.

plant biology↗

bioRxiv2023

Altered iron-sulfur cluster transfer in Arabidopsis mitochondria reveals lipoyl synthase as a Janus-faced enzyme that generates toxic sulfide

Iron-sulfur (Fe-S) cluster are vital cofactors in all domains of life. Mitochondrial Fe-S cluster assembly occurs in two major steps to first build [2Fe-2S] clusters and subsequently assemble these into [4Fe-4S] clusters. The two assembly machineries are interconnected by glutaredoxin S15 (GRXS15) that transfers [2Fe-2S] clusters to the second machinery. Diminished cluster transfer activity of GRXS15 in Arabidopsis mitochondria causes specific defects associated with lipoyl synthase (LIP1) activity. Conversely, overexpression of LIP1 in wild-type plants causes the release of toxic amounts of sulfide that can be detoxified by increasing the capacity for sulfide fixation through overexpression of O-acetylserine-(thiol)-lyase. The release of sulfide by lipoyl synthase causes a disturbance of mitochondrial sulfide homeostasis resulting in distinct and readily observable macroscopic phenotypes. These phenotypes enable a direct readout of consequences resulting from defects in Fe-S cluster assembly or targeted modulation of Fe-S cluster flux in mitochondria.

plant biology↗

bioRxiv2021

Endoplasmic reticulum oxidoreductin (ERO) provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. While many key players involved in oxidative protein folding are known, our understanding of how redox homeostasis in the ER is maintained and how EROs, the Cys residues of nascent proteins, and the luminal glutathione redox buffer interact is limited. Here, we isolated viable ero1 ero2 double mutants largely deficient in ERO activity, which rendered the mutants highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER lumen in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress, as induced by acute exposure to dithiothreitol, was delayed in ero1 ero2 mutants. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in the ero1 ero2 mutant reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis. One sentence summaryDynamic monitoring the ER luminal glutathione redox potential highlights the role of EROs in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress.

plant biology↗