An RK/ST C-Terminal Motif is Required for Targeting of OEP7.2 and a Subset of Other Arabidopsis Tail-Anchored Proteins to the Plastid Outer Envelope Membrane.

Tail-anchored (TA) proteins are a unique class of integral membrane proteins that possess a single C-terminal transmembrane domain and target post-translationally to the specific organelles at which they function. While significant advances have been made in recent years in elucidating the mechanisms and molecular targeting signals involved in the proper sorting of TA proteins, particularly to the endoplasmic reticulum and mitochondria, relatively little is known about the targeting of TA proteins to the plastid outer envelope. Here we show that several known or predicted plastid TA outer envelope proteins (OEPs) in Arabidopsis possess a C-terminal RK/ST sequence motif that serves as a conserved element of their plastid targeting signal. Evidence for this conclusion comes primarily from experiments with OEP7.2, which is a member of the Arabidopsis 7 kDa OEP family. We confirmed that OEP7.2 is localized to the plastid outer envelope and possesses a TA topology, and its C-terminal sequence (CTS), which includes the RK/ST motif, is essential for proper targeting to plastids. The CTS of OEP7.2 is functionally interchangeable with the CTSs of other TA OEPs that possess similar RK/ST motifs, but not with those that lack the motif. Further, a bioinformatics search based on a consensus sequence led to the identification of several new OEP TA proteins. Collectively, this study provides new insight into the mechanisms of TA protein sorting in plant cells, defines a new targeting signal element for a subset of TA OEPs and expands the number and repertoire of TA proteins at the plastid outer envelope.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.