The methylotrophic yeast, Pichia pastoris, is the most successful and favored microbial eukaryotic expression system for the production of recombinant proteins for biopharmaceutical or industrial purposes. P. pastoris has the ability to produce foreign proteins at high levels extracellularly, and since it secretes few endogenous proteins, this ability eliminates the need for expensive purification costs. It also combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most commonly utilized secretion signal leader in P. pastoris is the MATα prepro signal leader, originally found in S. cerevisiae. However, because some proteins cannot be secreted efficiently by P. pastoris, strategies to enhance secretion efficiency have involved the modification of the MATα prepro secretion signal leader. The study focuses on using site-directed mutagenesis of specific sets of amino acids of MATα prepro secretion leader to evaluate the correlation between secondary structure and secretion level. MATα pro-HRP mutants were created, in order to analyze the export of heterologous proteins in P. pastoris. In addition, structural analysis through circular dichroism was performed on mutant MATα pro-peptides to evaluate differences in secondary structure as a result of the mutagenesis. Mutants, pSC6 (Δ57-65) and pSC7 (Δ66-70) did not generate the same HRP secretion level as Δ57-70. In addition, a new proposed model of MATα pro-peptide signal leader was created. This new model suggests that the N and C terminus of MATα pro-peptide need to be presented correctly for proper interaction with secretion machinery and for efficient protein secretion. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most successful and commonly used secretion signal leader in Pichia pastoris has been the MAT α prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently even with the MAT α prepro secretion signal. Some strategies to enhance secretion efficiency involved modifying the secretion signal leader. Based on the knob-socket model and Jpred3 ( a secondary structure predictor), eleven deletions of MAT α prepro secretion signal and one MAT α pre double pro-peptide mutant was engineered and assayed with either horseradish peroxidase (HRP), or Candida antarctica lipase B reporter protein to evaluate the correlation between secondary structure and secretion level. In addition, structural analysis through circular dichroism was completed for the wild type pro-peptide and a mutant pro-peptide to evaluate differences in secondary structure. Results suggest pro-peptide amino acids 75-78 play an important role in determining secretion level and a higher secretion level tends to associate with secondary structures that are less defined. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

Pichia pastoris is a methylotrophic yeast that has been used in both research and industrial settings for recombinant protein expression due to the ease of genetically modifying its genome, its ability to grow to large densities in inexpensive media, and its capability to perform posttranslational modifications. Multiple tools such as the cis -acting factors MATα secretion signal and MBP fusion partners, and trans-acting modifications such as the bgs mutants have increased heterologous protein secretion. Although these techniques have already been used, their effects on the protein secretory pathway have yet to be elucidated. In this study, fluorescence microscopy was used to compare the localization of proteins expressed with the mutated MATα with the deletion of amino acids 57-70 to the wild type MATα secretion signal. Additional fluorescence microscopy was completed to visualize the localization of MBP-EGFP and EGFP-MBP fusion proteins and their spatial relativity to organelle markers. EGFP-MBP was used to further distinguish the properties of multiple bgs mutants. Additionally, secreted lipase activity levels were evaluated in bgs13 strains expressing either the wild type or the mutated MAT&agr; signal peptide. The results indicated that regardless of their differences, the MATα secretion signals and bgs mutants transported their cargo proteins through similar pathways within the cells. The results of the MBP fusion proteins suggest that the arrangement of MBP significantly influences protein secretion and localization. Lastly, the bgs13 mutant with MATα secretion signals demonstrated that lipase activity increased additively when cis- and trans -acting mutations were combined. Ultimately, these results can provide better understanding of each modified factor and the protein sorting pathway, leading to potential techniques that optimize protein secretion in P. pastoris .

This study investigates endocytosis and ER export signals of the yeast α-factor receptor and the role that receptor oligomerization plays in these processes. The α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, I found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays strongly suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, the recognition of endocytosis signal sequence and the recognition of the ligand-induced conformational change are likely to be two independent events, where the signal sequence plays only a passive role in the ligand-induced endocytosis. Four amino acid substitutions (C59R, H94P, S141P and S145P) in TM domains I, II and III were identified that resulted in the accumulation of truncated receptors in the ER but did not block ER export of full-length receptors. The two DXE motifs in the C-terminal tail were required for export of the mutant receptors from the ER; however DXE was not essential for proper cell surface expression of wild-type receptors apparently because the receptors contain redundant ER export signals. An assay for oligomerization of receptors in the ER was developed based on the ability of truncated mutant receptors to exit the ER. The four substitutions (C59R, H94P, S141P and S145P) that caused DXE-dependent ER export failed to form homo-oligomers, suggesting that the DXE motifs and receptor oligomerization serve as independent ER export signals. Consistent with this view, two of the substitutions (S141P and S145P), when coexpressed, with wild-type receptors, formed hetero-oligomers that exited the ER. Finally, the full-length oligomer-defective mutant Ste2-S141P was sensitive to α-factor, suggesting that receptor monomers that reach the cell surface are able to activate the heterotrimeric G protein. The potential roles that TM1, 2 and 3 play in receptor oligomerization are discussed.