Identification, characterization, and sub-cellular localization of protein disulfide isomerase (PDI) in arabidopsis thaliana

Ph.D. University of Hawaii at Manoa 2011.Includes bibliographical references.The traditional protein disulfide isomerase (PDI) catalyzes the formation, reduction, and isomerization of disulfide bonds of most secretory proteins in the endoplasmic reticulum (ER) of eukaryotes. PDI also serves as a redox-response regulator and chaperone. By modifying disulfide bonds, PDI changes the folded state and activity of its target proteins, which is ultimately important for normal cell metabolism, disease resistance, growth and development. In contrast to animal and yeast PDIs, little information is currently available on the characterization and function of PDIs in plants. The genome of the model plant, Arabidopsis, contains 12 different PDI genes. Some of these have traditional structures very closely related to yeast and mammalian PDIs. However, several differ in the presence of signal peptides, KDEL motifs and the number and positions of thioredoxin domains. In addition, three species of PDIs in plants have transmembrane domains. For PDI antisera generation, immunolocalization, and complementation studies, recombinant expression systems were developed for three PDIs fused to 6X-His to enable purification. For functional studies of PDIs, I mapped and selected homozygous T-DNA knockout lines of the pdi2 (SALK_115574 and SALK _017090), pdi5 (SALK_010645), and pdi8 (SALK _023055) genes by using PCR. The absence of PDI mRNA and proteins were confirmed using RT-PCR and immunoblot analysis. To study protein-protein interactions of PDIs, I identified interacting partners of PDI2 and PDI5 using the yeast two-hybrid system and confirmed their interaction by co-immunoprecipitation, fluorescence resonance energy transfer (FRET), and β-galactosidase assays. PDI5 interacted with three different cysteine protease that played roles in programmed cell death (PCD). PDI2 interacted with several proteins in the secretory pathway as well as nuclear proteins, Rho and MEE8, both of which are transcription factors. To identify the sub-cellular localization of PDIs, PDI2 and PDI8 were transiently expressed as fusion proteins with green fluorescent protein (GFP) in leaf protoplasts. Co-expression with subcellular markers fused to RFP determined that PDI8 localized to the ER and was exported to the vacuole membrane (tonoplast). PDI2 was localized to the ER, Golgi, nucleus, and vacuole. The localizations of PDI2 were confirmed by immunolabeling and transmission electron microscopy