Poster: Organelle biogenesis

Abs # 631: Subcellular sorting pathways and molecular targeting signals for peroxisomal monodehydroascorbate reductase isoforms in Arabidopsis thaliana

Presenter:	Lingard, Matthew J., matthew.lingard@asu.edu
Authors	Lingard, Matthew J. (A)   Trelease, Richard N. (A)   Lisenbee, Cayle S (B)
Affiliations:	(A): Arizona State University (B): Mayo Clinic in Scottsdale

Genes coding for five different monodehydroascorbate reductase (MDAR) polypeptides have been identified in the Arabidopsis genome. A 53 kD form is present in mitochondria and chloroplasts. Two 47 kD forms (AtMDAR47b & c) may be cytosolic. Two forms appear to be in peroxisomes: AtMDAR47a in the matrix with the C-terminal –AKI-COOH putative PTS1 and AtMDAR54 in the membrane (Pl Physiol Abst 227, 2002) with a putative membrane PTS. Here, we determined the in vivo subcellular localizations of AtMDAR47a, b & c and partially characterized the targeting signals on peroxisomal MDARs. (Immuno)fluorescence microscopy of myc-tagged and GFP-linked MDARs transiently expressed in Arabidopsis and BY-2 suspension cells was employed. myc-AtMDAR47a sorted inefficiently to the peroxisomes; myc fluorescence was also seen in the cytosol. Pea myc-MDAR47, with a similar PTS1 (-SKI-COOH), also sorted inefficiently to peroxisomes; mutation to the canonical –SKL-COOH resulted in increased targeting efficiency in BY-2 but not Arabidopsis. Removal of –AKI-COOH from AtMDAR47a showed that it was necessary for targeting. myc-AtMDAR47b & c remained in the cytosol. myc-AtMDAR54, with a predicted transmembrane domain (TMD) nearby a C-terminal cluster of five basic amino acid residues sorted directly to peroxisomes; the basic residues were necessary for targeting. The C-terminal 38 amino acid residues (TMD-RRRRRW-COOH) were sufficient to target GFP to peroxisomes, whereas –RRRRRW-COOH or the TMD separately were not. In conclusion, AtMDAR47a is a peroxisomal matrix MDAR with a relatively inefficient PTS1, while AtMDAR47b & c are cytosolic forms. AtMDAR54 targets directly to peroxisomes via a C-terminal mPTS composed of a TMD adjacent to a cluster of five basic amino acid residues. NSF grant MCB-0091826.