Project Summary Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that has been genetically engineered to express heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In the past 25 years, over 5,000 proteins from bacteria to humans have been produced in this yeast. Despite the success of the P. pastoris expression system relatively little is known about the exact machinery and mechanisms behind its export of protein. The experiments in this proposal are aimed at elucidating a mechanistic understanding of the trans-acting factors involved in secretion. A small collection of genomic disruption mutants involved in secretion have been generated. Most of the twelve BGS (beta-galactosidase supersecretion) genes identified appear to have functions in intracellular signaling or vesicle transport. One particular mutant strain, bgs13, showed enhanced secretion of most recombinant proteins that were tested. The predicted amino acid sequence of Bgs13p is 50% identical and 68% similar to protein kinase C from the yeast Saccharomyces cerevisiae. This grant aspires to clarify how this mutant changes the efficiency of secretion by modification of the pathway and/or cellular architecture. Additionally, the structural qualities of both the mutant bgs13 protein and recombinant proteins expressed by the mutant bgs13 strain will be explored. Specific Aim 1 proposes to determine whether the supersecretion phenotype of the mutant bgs13 strain is due to a decrease in protein kinase C activity or a change in localization of mutant protein. Mass spectrometry will be used to examine the structural characteristics of both the mutant protein and recombinant proteins expressed in the bgs13 strain. Specific Aim 2 intends to determine whether the mutant supersecreting strain of Pichia pastoris, bgs13, changes the cell wall integrity, alters the glycosylation process,or impacts the unfolded protein response of the yeast. These effects can influence enhanced secretion or result in a cell wall defect. The illumination of secretory mechanisms will increase the basic understanding of how Pichia pastoris targets its proteins and the limitations of the secretory pathway. Additionally, these studies will aid the design of a better host system for scientists who use recombinant protein expression.