My thesis is focused upon molecularly defining the mechanism of early steps in metazoan nuclear pore assembly. Nuclear pore complexes (NPCs) are large proteinaceous structures that span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm. In metazoans, the massive NPCs disassemble into soluble subunits at the beginning of mitosis and then somehow reassemble following chromosome segregation; a process that is coordinated with membrane recruitment and fusion. The mechanism and order of NPC assembly is poorly understood. In Chapter 1, I show that ELYS co-purifies with the Nup107-160 complex, the largest subunit of the NPC, in Xenopus egg and human cell extracts. Indeed, I demonstrate that ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division. In Chapter 2, I focus on defining the early steps in nuclear pore assembly, including the mechanism for ELYS as the pore 'targeting' protein. In Chapter 3, I collaborated with Dr. Corinne Ramos on a study to order pore assembly with respect to inner and outer nuclear membrane fusion. Taken the data from chapters 1-3 together, I suggest a model in which NPC assembly is initiated on AT -rich chromatin through an interaction with the C-terminus of ELYS. The data also show the chromatin binding of ELYS precedes and is required for the binding of the Nup107-160 complex. Chromatin-bound ELYS and the Nup107-160 complex then recruit integral pore membrane proteins POM121- and NDC1-containing membrane vesicles. Membrane vesicle fusion takes place to form patches of continuous double nuclear membranes. Oligomerization of ELYS/Nup107-160/POM121 then acts to promote fusion between the inner and outer nuclear membranes to form a diffusion channel. Finally, the remaining soluble pore subunits are recruited to assemble the mature, functional nuclear pore. Finally, Appendix B presents data on the Xenopus binding partners of the C- terminus of Nup160, a member of the Nup107-160 complex, which was derived from mass spectrometry analyses. This data led to the discovery that vertebrate centrin 2 localizes to NPCs and functions in mRNA and protein export, as described in Chapter 4

Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a "plant-like" algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN.

Transgenic microalgae have the potential to impact many diverse biotechnological industries including energy, human and animal nutrition, pharmaceuticals, health and beauty, and specialty chemicals. However, major obstacles to sophisticated genetic and metabolic engineering in algae have been the lack of well-characterized transformation vectors to direct engineered gene products to specific subcellular locations, and the inability to robustly express multiple nuclear-encoded transgenes within a single cell. Here we validate a set of genetic tools that enable protein targeting to distinct subcellular locations, and present two complementary methods for multigene engineering in the eukaryotic green microalga Chlamydomonas reinhardtii. The tools described here will enable advanced metabolic and genetic engineering to promote microalgae biotechnology and product commercialization.