Subcellular localization, sorting, and targeting signals of Arabidopsis peroxin 16

Peroxin 16 homologs have been observed only in three organisms namely Homo sapiens, Yarrowia lipolytica and Arabidopsis thaliana. The localizations, sorting pathways and the molecular targeting signals of Arabidopsis thaliana peroxin 16 homolog (AtPex16p) were examined in this dissertation. Results of Chapter 1 aimed at finding the localization of AtPex16p in Arabidopsis cells revealed that endogenous AtPex16p coexists in endoplasmic reticulum (ER) and peroxisomes at steady state. Cell fractionation experiments revealed that AtPex16p behaved as a peripheral membrane protein and topologically was facing the cytosol in ER and the matrix in peroxisomes. In Chapter 2, emphasis was given to the sorting pathways of AtPex16p in Arabidopsis and BY-2 cells. Transiently-expressed mycAtPex16p sorted indirectly to pre-existing peroxisomes from ER via pre-peroxisomel vesicles (PPV) in both Arabidopsis and BY-2 cells. These conclusions were reached from results of time-course experiments, brefeldinA (BFA) treatments and cold incubations of cells at 15°C obtained via immunofluorescence microscopy. In addition, AtPex16p was found to sort to pre-existing peroxisomes from ER via PPV distinct from the ER-derived ascorbate peroxidase (APX) transporting vesicles. In the third chapter, molecular targeting signals necessary and sufficient to target transiently-expressed AtPex16p from the cytosol to the ER and from the ER to pre-existing peroxisomes were identified. Site-specific amino acid substitutions and/or deletions within transiently expressed mycAtPex16p were employed and the results were analyzed via immunofluorescence microscopy. A basic cluster of internal amino acid residues adjacent to two membrane helices (MH) were necessary for targeting mycAtPex16p to ER and then to peroxisomes. These residues, however, were not sufficient for targeting nascent chimeric green fluorescent protein (GFP) to ER. These basic residues along with two adjacent MH were sufficient for targeting and sorting chimeric GFP to ER and on to peroxisomes in both the cell types. Moreover, the signals necessary and sufficient for targeting to these two organellar compartments were overlapping and hence non-distinguishable. Thus, the conclusions derived from this dissertation research provide important insight into the involvement of ER in trafficking of an authentic plant peroxin.