(C.P.L.G., J.M.P.) AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K 0.5 5 0.066 6 0.025 mM). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and a-methyl-Dglucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to b-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition.

Summary The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small Translocase of the Inner Membrane (Tim) proteins and cysteine rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, MitoBloCK-6, attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the Translocase of the Outer Membrane (TOM) complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6 exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.

Significance The lethal disorder, primary hyperoxaluria 1 (PH1), is caused by mutations in peroxisomal-localized alanine:glyoxylate aminotransferase (AGT). AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate a strong N-terminal mitochondrial targeting sequence that directs AGT to mitochondria. Although mutant AGT is functional, the enzyme must be in the peroxisome to detoxify glyoxylate and prevent oxalate accumulation. We have identified a Food and Drug Administration-approved drug, dequalinium chloride (DECA), from a chemical genetic screen to identify probes that attenuate mitochondrial protein import. DECA treatment restores trafficking of mutant AGT from mitochondria to peroxisomes with a subsequent reduction in oxalate levels. Thus, repurposing DECA has potential in therapeutic strategies for PH1 because current clinical trials have not produced an effective treatment, short of organ transplant.