Moonlighting proteins: putting the spotlight on enzymes

Abstract

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.

Keywords: Arc mutants; FtsZ; arogenate dehydratase; chloroplast division; gene families; moonlighting proteins; phenylalanine biosynthesis.

Figure 1.

ADT2 and chloroplast morphology. A. Confocal 3D image of a WT chloroplast expressing ADT2-CFP. ADT2-CFP forms a ring at the equatorial plane of dividing chloroplasts. B. TEM image of a chloroplast in adt2-1D. Chloroplasts in the Arabidopsis adt2 mutant line are often larger and misshapen.

Figure 2.

Expression of ADT2 and FtsZ2 in wildtype and arc mutants. A. ADT2 and FtsZ2 were transiently expressed as ADT2-CFP or FtsZ2-YFP fusion proteins in A. thaliana wildtype and adt2-1D mutant leaves. Expression of wild type ADT2 in the adt2-1D background essentially complements the mutant while FtsZ2 is misexpressed. Shown are merges of chlorophyll fluorescence and ADT2-CFPs (left) and FtsZ2-YFP (right). B. Co-expression of ADT2-CFP and FtsZ2-YFP in N. benthamiana leaves. Both proteins co-localize as rings at the equatorial plane of dividing chloroplasts. Shown are all individual channels image (CFP: top left, YFP: top right, chlorophyll: bottom left) and the merged (bottom right) where co-localization of YFP and CFP is seen as green. C. ADT2 and FtsZ2 expression patterns in three A. thaliana chloroplast division mutants (arc3-1, arc5-1 and arc6-1). Two samples of ADT2 expression are shown (left and middle column) and compared to FtsZ2 expression (right column). In arc3 and arc6 mutants, ADT2 is misexpressed, and expression patterns resemble that of FtsZ2 in the same mutants. In arc5, ADT2 is often seen as a single ring in both enlarged and chloroplasts that are wild type in size (arrows). Occasionally multiple rings are observed coinciding with constriction points. Merged images of chlorophyll autofluorescence and ADT2-CFPs (left and middle) and FtsZ2-YFP (right).

Figure 3.

Proposed role of ADT2 in chloroplast division. The placement of chloroplast division rings is a highly co-ordinated process where the inner two rings are anchored first inside of the inner membrane with the help of ARC6 and PARC6. This is followed by the anchoring of the outer PD ring and then the ARC5 (dynamin) ring on the outside of the outer membrane. Anchoring of the outer rings requires the function of PDV1 and PDV2. Based on our data we propose that ADT2 is forming one of the inner rings either (A) coinciding with establishing the Z-ring, formed by the FtsZ1 and FtsZ2 heterodimer or (B) preceding and required for the formation of the Z-ring.