Moonlighting proteins: putting the spotlight on enzymes.

AROGENATE DEHAYDRATASE2 (ADT2) is a member of the Arabidopsis thaliana ADT family. All members of this family act as arogenate dehydratases in phenylalanine biosynthesis, decarboxylating/dehydrating arogenate to phenylalanine. ADT2 is detected in stromules, and as a ring around the equatorial plane of dividing chloroplasts, indicating it has a second, non-enzymatic function in chloroplast division. Here, we provide further evidence for this alternative role of ADT2. First, we demonstrate that ADT2 and FtsZ co-localize around the equatorial plane at the same time. Second, FtsZ expression in an adt2 mutant was analyzed, as well as ADT2 expression in three Arabidopsis chloroplast division mutants, ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), ARC5 and ARC6. In arc3 and arc6 mutants, ADT2 is misexpressed and resembles the expression of FtsZ in the same mutants. However, in the arc5 mutant, ADT2 ring positioning is observed at constriction points indicating proper relative timing. ADT2 expression in the arc mutants shows that the role of ADT2 in chloroplast division occurs prior to ARC5, but is dependent on ARC3 and ARC6. Abbreviations used: ADT: arogenate dehydratase, ARC: accumulation and replication of chloroplasts, CFP: cyan fluorescent protein, dpi: days post infiltration, FtsZ: filamentous temperature sensitive Z, PD: plastid division, Phe: phenylalanine, YFP: yellow fluorescent protein.

Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles.

Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively.