Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Susanne Kohalmi


Arogenate dehydratases (ADTs) have been identified to catalyze the last step of phenylalanine (Phe) biosynthesis in plants. All ADTs have a transit peptide sequence that targets them into the chloroplasts where the biosynthesis of Phe happens. Subcellular localization studies using fluorescently tagged Arabidopsis thaliana ADTs demonstrated that all six ADTs localize to chloroplast stromules (stroma filled tubules). However, one member of this family, ADT5, was also detected in the nucleus. As dual targeting of proteins to different cell compartments is an indication of multifunctionality, ADT5 nuclear localization suggests that this member of the ADT protein family is a moonlighting protein with a non-enzymatic role in the nucleus.

In this study, first the nuclear localization of the ADT5 was confirmed by expression of ADT5-CFP under the regulation of ADT5 native promoter. Using confocal microscopy and Western blot analysis it was shown that ADT5 localizes into the nucleus. Next, different possible mechanisms that could result to the nuclear localization of ADT5 were studied. It was tested if ADT5 can move directly from chloroplast stroma to the nucleus through stromules or if ADT5 enter the nucleus from cytoplasm using the nuclear import system. Data presented are consistent with a translocation from cytoplasm. A combination of an AQEH motif and a single amino acid Asn28 present in the N-terminus of ADT5 ACT domain were identified as potential protein interaction sites required for nuclear targeting of ADT5. Y2H screenings and protein-protein interaction analyses suggest that nuclear localization of ADT5 occurs through the interaction of the cytosolic portion of an ER membrane bound protein, PHOSPHOLIPID DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1). A nuclear targeting sequence was identified in the N-terminal cytosolic portion of PDAT1. Hence, it is possible that PDAT1 piggybacks ADT5 into the nucleus through the nuclear import system. This study introduces ADT5 as a moonlighting protein and identifies a possible mechanism for ADT5 nuclear translocation.