Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Dr. Rima Menassa

2nd Supervisor

Dr. Susanne Kohalmi

Joint Supervisor

Abstract

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles found in seeds whose function is to accumulate seed storage proteins. It was shown that PB formation is not limited to seeds, and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP), hydrophobin-I (HFBI) or Zera® fusion tags induces PBs in leaves of Nicotiana benthamiana. The mechanism by which fusion tags induce PBs is not well understood. To address how PBs form and develop in plant leaves, I studied the factors involved in their formation including recombinant protein concentration, effect of the fusion tags, PB sequestration patterns and destination in vivo.

In this study I showed that PB formation is a concentration-dependent mechanism and that proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. The presence of fusion tags is not necessary for the formation of PBs but affects their distribution pattern and size. ELP-induced PBs are larger than HFBI-induced PBs and the size of both PBs increases over time along with accumulation levels of the recombinant protein. I found that in the process of PB formation, secretory and ER resident molecules are passively sequestered into the lumen of PBs. This property of PBs was proposed as a tool to increase accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins. To understand whether PBs are terminally-stored cytosolic organelles or if they are connected to the ER and to each other, I developed an EGFP-based photoconversion technique, which was successfully used to visualize the trafficking of proteins targeted to the cytosol, ER, apoplast, and chloroplast in vivo. Study of PBs with this technique suggested that PBs remain associated with the ER and communicate with one another via the ER.

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Supplementary Movie - 4.6

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Supplementary Movie - 4.7


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