Abstract 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 statement We 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.

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